{"format":"molecular-machines.cards","version":1,"updated":"2026-10-01","decks":[{"machine":"atp-synthase","name":"ATP synthase","count":28,"url":"/learn/cards/atp-synthase.json"},{"machine":"flagellar-motor","name":"Bacterial flagellar motor","count":27,"url":"/learn/cards/flagellar-motor.json"},{"machine":"kinesin","name":"Kinesin","count":28,"url":"/learn/cards/kinesin.json"},{"machine":"myosin","name":"Myosin","count":28,"url":"/learn/cards/myosin.json"},{"machine":"dynein","name":"Dynein","count":28,"url":"/learn/cards/dynein.json"},{"machine":"ribosome","name":"Ribosome","count":28,"url":"/learn/cards/ribosome.json"},{"machine":"rna-polymerase","name":"RNA polymerase II","count":28,"url":"/learn/cards/rna-polymerase.json"},{"machine":"groel","name":"GroEL–GroES","count":28,"url":"/learn/cards/groel.json"},{"machine":"proteasome","name":"Proteasome","count":28,"url":"/learn/cards/proteasome.json"},{"machine":"serca","name":"SERCA calcium pump","count":28,"url":"/learn/cards/serca.json"},{"machine":"hemoglobin","name":"Hemoglobin","count":28,"url":"/learn/cards/hemoglobin.json"},{"machine":"cas9","name":"Cas9","count":28,"url":"/learn/cards/cas9.json"},{"machine":"shared","name":"Across machines","count":20,"url":"/learn/cards/shared.json"}],"cards":[{"id":"atp-synthase-energy-source","machine":"atp-synthase","kind":"cloze","prompt":"ATP synthase in mitochondria is driven by the {{proton-motive force}} across the inner membrane.","answer":"proton-motive force","explanation":"Protons flow back down their gradient through the machine. Some bacteria use a sodium-motive force instead.","section":"summary","topic":"purpose","sources":["machine:energy","machine:where"],"tags":["energy","protons"],"difficulty":1,"url":"/machines/atp-synthase#summary","cites":[{"source":"machine:energy","machine":"atp-synthase","label":"Summary","section":"summary"},{"source":"machine:where","machine":"atp-synthase","label":"Summary","section":"summary"}]},{"id":"atp-synthase-ring-of-c-subunits","machine":"atp-synthase","kind":"cloze","prompt":"In ATP synthase, protons crossing the membrane spin a ring of {{c subunits}}.","answer":"c subunits","explanation":"Each c subunit carries one proton site on a conserved carboxylate, so the c-ring turns as sites load and unload.","section":"summary","topic":"parts","sources":["machine:summary","component:c-ring"],"tags":["c-ring","rotation"],"difficulty":1,"url":"/machines/atp-synthase#summary","cites":[{"source":"machine:summary","machine":"atp-synthase","label":"Summary","section":"summary"},{"source":"component:c-ring","machine":"atp-synthase","label":"Part: c-ring","section":"summary"}]},{"id":"atp-synthase-gamma-role","machine":"atp-synthase","kind":"qa","prompt":"Which ATP synthase subunit turns inside the α3β3 head and sets the state of each catalytic β subunit?","answer":"γ, the central stalk.","explanation":"The c-ring carries γ with it, so γ links the membrane rotor to the three catalytic sites in the head.","section":"summary","topic":"parts","sources":["component:γ","mechanism:The rotor turns the central stalk","ref:noji1997"],"tags":["gamma","rotation"],"difficulty":1,"url":"/machines/atp-synthase#summary","cites":[{"source":"component:γ","machine":"atp-synthase","label":"Part: γ","section":"summary"},{"source":"mechanism:The rotor turns the central stalk","machine":"atp-synthase","label":"Step: The rotor turns the central stalk","section":"mechanism"},{"source":"ref:noji1997","machine":"atp-synthase","label":"Noji et al.","section":"sources","anchor":"ref-noji1997","href":"https://doi.org/10.1038/386299a0"}]},{"id":"atp-synthase-atp-per-turn","machine":"atp-synthase","kind":"cloze","prompt":"One full turn of the ATP synthase rotor makes {{3}} ATP, one per β subunit.","answer":"3","explanation":"There are three catalytic β subunits, and each 120° turn of γ releases one ATP.","section":"summary","topic":"numbers","sources":["stat:ATP per full turn","ref:watt2010"],"tags":["atp","numbers"],"difficulty":1,"url":"/machines/atp-synthase#summary","cites":[{"source":"stat:ATP per full turn","machine":"atp-synthase","label":"Key number: ATP per full turn","section":"summary"},{"source":"ref:watt2010","machine":"atp-synthase","label":"Watt et al.","section":"sources","anchor":"ref-watt2010","href":"https://doi.org/10.1073/pnas.1011099107"}]},{"id":"atp-synthase-c-ring-range","machine":"atp-synthase","kind":"cloze","prompt":"Across species, the ATP synthase c-ring has {{8 to 15}} c subunits.","answer":"8 to 15","explanation":"It runs from c8 in animal mitochondria to c15 in Spirulina. A bigger ring costs more protons per ATP.","section":"summary","topic":"numbers","sources":["stat:c-ring size across species","evolution:Ring size as gear ratio","ref:watt2010"],"tags":["c-ring","species"],"difficulty":2,"url":"/machines/atp-synthase#summary","cites":[{"source":"stat:c-ring size across species","machine":"atp-synthase","label":"Key number: c-ring size across species","section":"summary"},{"source":"evolution:Ring size as gear ratio","machine":"atp-synthase","label":"Ring size as gear ratio","section":"summary"},{"source":"ref:watt2010","machine":"atp-synthase","label":"Watt et al.","section":"sources","anchor":"ref-watt2010","href":"https://doi.org/10.1073/pnas.1011099107"}]},{"id":"atp-synthase-bedaquiline","machine":"atp-synthase","kind":"qa","prompt":"How does the tuberculosis drug bedaquiline stop the mycobacterial ATP synthase?","answer":"It jams the c-ring, so the rotor stalls.","explanation":"It treats multidrug-resistant tuberculosis. Structures of the drug on the bacterial and human enzymes now guide safer analogs.","section":"summary","topic":"debate","sources":["frontier:Drugs that jam the rotor","species:Mycobacteria","ref:preiss2015","ref:andries2005"],"tags":["drug","c-ring"],"difficulty":2,"url":"/machines/atp-synthase#summary","cites":[{"source":"frontier:Drugs that jam the rotor","machine":"atp-synthase","label":"Open question: Drugs that jam the rotor","section":"summary"},{"source":"species:Mycobacteria","machine":"atp-synthase","label":"Mycobacteria","section":"summary"},{"source":"ref:preiss2015","machine":"atp-synthase","label":"Preiss et al.","section":"sources","anchor":"ref-preiss2015","href":"https://doi.org/10.1126/sciadv.1500106"},{"source":"ref:andries2005","machine":"atp-synthase","label":"Andries et al.","section":"sources","anchor":"ref-andries2005","href":"https://doi.org/10.1126/science.1106753"}]},{"id":"atp-synthase-custom-ratio-use","machine":"atp-synthase","kind":"qa","prompt":"Why would an engineered ATP synthase with a higher H+/ATP ratio (up to 5.8) be useful?","answer":"It could make ATP at proton gradients too weak for natural enzymes.","explanation":"Point mutations change the c-ring size, and extra peripheral stalks raise H+/ATP to 5.8. This work is still lab-scale.","section":"summary","topic":"debate","sources":["frontier:Custom proton-to-ATP ratios","ref:ueno2025","ref:pogoryelov2012"],"tags":["engineering","h-per-atp"],"difficulty":2,"url":"/machines/atp-synthase#summary","cites":[{"source":"frontier:Custom proton-to-ATP ratios","machine":"atp-synthase","label":"Open question: Custom proton-to-ATP ratios","section":"summary"},{"source":"ref:ueno2025","machine":"atp-synthase","label":"Ueno et al.","section":"sources","anchor":"ref-ueno2025","href":"https://doi.org/10.1038/s41467-025-61227-w"},{"source":"ref:pogoryelov2012","machine":"atp-synthase","label":"Pogoryelov et al.","section":"sources","anchor":"ref-pogoryelov2012","href":"https://doi.org/10.1073/pnas.1120027109"}]},{"id":"atp-synthase-arginine-barrier","machine":"atp-synthase","kind":"qa","prompt":"Why can a proton not slip straight across subunit a of ATP synthase?","answer":"The two half-channels in subunit a do not meet, and a conserved arginine sits between them.","explanation":"So the only way through is to ride on the c-ring almost a full turn, from one half-channel to the other.","section":"mechanism","topic":"cycle","sources":["step:Protons cross subunit a","mechanism:The proton leaves on the other side","ref:allegretti2015"],"tags":["subunit-a","protons"],"difficulty":2,"url":"/machines/atp-synthase#mechanism","cites":[{"source":"step:Protons cross subunit a","machine":"atp-synthase","label":"Step: Protons cross subunit a","section":"mechanism"},{"source":"mechanism:The proton leaves on the other side","machine":"atp-synthase","label":"Step: The proton leaves on the other side","section":"mechanism"},{"source":"ref:allegretti2015","machine":"atp-synthase","label":"Allegretti et al.","section":"sources","anchor":"ref-allegretti2015","href":"https://doi.org/10.1038/nature14185"}]},{"id":"atp-synthase-neutral-site-enters-lipid","machine":"atp-synthase","kind":"qa","prompt":"Why can only a protonated site on the ATP synthase c-ring turn into the membrane lipid?","answer":"A neutral carboxylate can enter the oily core of the membrane; a charged one cannot.","explanation":"Thermal motion jiggles the ring, and this charge rule lets only forward turns stick. The proton gradient pays for that bias.","section":"mechanism","topic":"cycle","sources":["step:The c-ring turns one step","mechanism:The c-ring glutamate takes the proton","ref:meier2005"],"tags":["c-ring","protons"],"difficulty":2,"url":"/machines/atp-synthase#mechanism","cites":[{"source":"step:The c-ring turns one step","machine":"atp-synthase","label":"Step: The c-ring turns one step","section":"mechanism"},{"source":"mechanism:The c-ring glutamate takes the proton","machine":"atp-synthase","label":"Step: The c-ring glutamate takes the proton","section":"mechanism"},{"source":"ref:meier2005","machine":"atp-synthase","label":"Meier et al.","section":"sources","anchor":"ref-meier2005","href":"https://doi.org/10.1126/science.1111199"}]},{"id":"atp-synthase-binding-change","machine":"atp-synthase","kind":"cloze","prompt":"In ATP synthase, each 120° turn of γ moves every β subunit {{one state along}}.","answer":"one state along","explanation":"At any moment one β is open and empty and two are closed. This is Boyer's binding-change mechanism.","section":"mechanism","topic":"cycle","sources":["step:Each β changes shape","mechanism:Each β cycles through three states","ref:boyer1997"],"tags":["beta","binding-change"],"difficulty":2,"url":"/machines/atp-synthase#mechanism","cites":[{"source":"step:Each β changes shape","machine":"atp-synthase","label":"Step: Each β changes shape","section":"mechanism"},{"source":"mechanism:Each β cycles through three states","machine":"atp-synthase","label":"Step: Each β cycles through three states","section":"mechanism"},{"source":"ref:boyer1997","machine":"atp-synthase","label":"Boyer, Annu Rev Biochem 1997","section":"sources","anchor":"ref-boyer1997","href":"https://doi.org/10.1146/annurev.biochem.66.1.717"}]},{"id":"atp-synthase-tight-site","machine":"atp-synthase","kind":"cloze","prompt":"In ATP synthase, a β site takes up ADP and phosphate while open, and ATP forms when the site closes {{tightly}}.","answer":"tightly","explanation":"Open, loose and tight follow each other as γ turns 120° at a time. The next opening releases the ATP.","section":"mechanism","topic":"cycle","sources":["step:ATP forms and leaves","mechanism:Each β cycles through three states","ref:boyer1997"],"tags":["beta","binding-change"],"difficulty":2,"url":"/machines/atp-synthase#mechanism","cites":[{"source":"step:ATP forms and leaves","machine":"atp-synthase","label":"Step: ATP forms and leaves","section":"mechanism"},{"source":"mechanism:Each β cycles through three states","machine":"atp-synthase","label":"Step: Each β cycles through three states","section":"mechanism"},{"source":"ref:boyer1997","machine":"atp-synthase","label":"Boyer, Annu Rev Biochem 1997","section":"sources","anchor":"ref-boyer1997","href":"https://doi.org/10.1146/annurev.biochem.66.1.717"}]},{"id":"atp-synthase-energy-goes-to-release","machine":"atp-synthase","kind":"qa","prompt":"In ATP synthase, which part of the catalytic cycle uses most of the proton energy?","answer":"Releasing the finished ATP from the site.","explanation":"The proton-motive force is used mainly to release ATP from the site, not to form it. The turning stalk pries the new ATP loose.","section":"mechanism","topic":"cycle","sources":["fact:A26","stop:site","ref:boyer1998"],"tags":["energy","binding-change"],"difficulty":3,"url":"/machines/atp-synthase#mechanism","cites":[{"source":"fact:A26","machine":"atp-synthase","label":"Where the proton energy goes: Most into releasing ATP","section":"story"},{"source":"stop:site","machine":"atp-synthase","label":"Big picture: Catalytic site","section":"story"},{"source":"ref:boyer1998","machine":"atp-synthase","label":"Boyer 1998","section":"sources","href":"https://doi.org/10.1002/(SICI)1521-3773(19980918)37:17<2296::AID-ANIE2296>3.0.CO;2-W"}]},{"id":"atp-synthase-symmetry-mismatch","machine":"atp-synthase","kind":"qa","prompt":"How can an 8-subunit c-ring drive an ATP synthase head that works in three 120° steps?","answer":"The stalks and the head flex to absorb the mismatch.","explanation":"The rotor and stalk store elastic energy between proton steps. E. coli structures show the peripheral stalk bending and twisting as the rotor moves.","section":"mechanism","topic":"cycle","sources":["step:γ turns inside the head","mechanism:The rotor turns the central stalk","analogy:A loaded spring","ref:sobti2020"],"tags":["elastic","c-ring"],"difficulty":3,"url":"/machines/atp-synthase#mechanism","cites":[{"source":"step:γ turns inside the head","machine":"atp-synthase","label":"Step: γ turns inside the head","section":"mechanism"},{"source":"mechanism:The rotor turns the central stalk","machine":"atp-synthase","label":"Step: The rotor turns the central stalk","section":"mechanism"},{"source":"analogy:A loaded spring","machine":"atp-synthase","label":"Analogy: A loaded spring","section":"story"},{"source":"ref:sobti2020","machine":"atp-synthase","label":"Sobti et al.","section":"sources","anchor":"ref-sobti2020","href":"https://doi.org/10.1038/s41467-020-16387-2"}]},{"id":"atp-synthase-reverse-mode","machine":"atp-synthase","kind":"qa","prompt":"What does ATP synthase do when ATP is present but the proton gradient is weak?","answer":"It runs backward: F1 drives the rotor the other way and Fo pumps protons.","explanation":"In mitochondria, the inhibitor protein IF1 blocks this reverse mode.","section":"mechanism","topic":"cycle","sources":["mechanism:The motor runs both ways","component:IF1","ref:noji1997","ref:diez2004"],"tags":["reverse","if1"],"difficulty":2,"url":"/machines/atp-synthase#mechanism","cites":[{"source":"mechanism:The motor runs both ways","machine":"atp-synthase","label":"Step: The motor runs both ways","section":"mechanism"},{"source":"component:IF1","machine":"atp-synthase","label":"Part: IF1","section":"summary"},{"source":"ref:noji1997","machine":"atp-synthase","label":"Noji et al.","section":"sources","anchor":"ref-noji1997","href":"https://doi.org/10.1038/386299a0"},{"source":"ref:diez2004","machine":"atp-synthase","label":"Diez et al.","section":"sources","anchor":"ref-diez2004","href":"https://doi.org/10.1038/nsmb718"}]},{"id":"atp-synthase-n-over-3","machine":"atp-synthase","kind":"qa","prompt":"Why does an ATP synthase with a c-ring of n subunits spend n/3 protons per ATP?","answer":"One full turn moves n protons, one per c subunit, and makes 3 ATP.","explanation":"So animals (c8) pay 2.7 protons per ATP, yeast (c10) 3.3 and chloroplasts (c14) 4.7.","section":"mechanism","topic":"cycle","sources":["mechanism:One turn costs n protons","ref:watt2010"],"tags":["h-per-atp","c-ring"],"difficulty":2,"url":"/machines/atp-synthase#mechanism","cites":[{"source":"mechanism:One turn costs n protons","machine":"atp-synthase","label":"Step: One turn costs n protons","section":"mechanism"},{"source":"ref:watt2010","machine":"atp-synthase","label":"Watt et al.","section":"sources","anchor":"ref-watt2010","href":"https://doi.org/10.1073/pnas.1011099107"}]},{"id":"atp-synthase-body-weight-per-day","machine":"atp-synthase","kind":"cloze","prompt":"A person remakes about {{their own body weight}} of ATP every day, most of it made by ATP synthase.","answer":"their own body weight","explanation":"An estimate from oxygen use: 54–68 kg a day for a college student.","section":"story","topic":"purpose","sources":["fact:A2","stop:body","story:summary","ref:bnid105606"],"tags":["atp","body"],"difficulty":1,"url":"/machines/atp-synthase#story","cites":[{"source":"fact:A2","machine":"atp-synthase","label":"ATP remade per day: 1 body weight","section":"story"},{"source":"stop:body","machine":"atp-synthase","label":"Big picture: You","section":"story"},{"source":"story:summary","machine":"atp-synthase","label":"Big picture","section":"story"},{"source":"ref:bnid105606","machine":"atp-synthase","label":"BNID 105606","section":"sources","href":"https://bionumbers.hms.harvard.edu/bionumber.aspx?id=105606"}]},{"id":"atp-synthase-heart-stock","machine":"atp-synthase","kind":"qa","prompt":"Why must the ATP synthases of the heart make ATP as fast as the heart uses it?","answer":"The heart keeps only a few seconds' worth of ATP in stock.","explanation":"Yet the heart cycles about 6 kg of ATP a day, an estimate from a review.","section":"story","topic":"purpose","sources":["fact:A6","stop:heart","fact:A4","ref:grynberg1996"],"tags":["heart","atp"],"difficulty":1,"url":"/machines/atp-synthase#story","cites":[{"source":"fact:A6","machine":"atp-synthase","label":"The heart's ATP store: a few seconds of beating","section":"story"},{"source":"stop:heart","machine":"atp-synthase","label":"Big picture: Heart","section":"story"},{"source":"fact:A4","machine":"atp-synthase","label":"ATP cycled by the heart: 6 kg per day","section":"story"},{"source":"ref:grynberg1996","machine":"atp-synthase","label":"Grynberg 1996","section":"sources","href":"https://doi.org/10.1097/00005344-199600003-00003"}]},{"id":"atp-synthase-atp-per-second","machine":"atp-synthase","kind":"cloze","prompt":"One mitochondrial ATP synthase turning about 100 times per second makes about {{300}} ATP per second.","answer":"300","explanation":"3 ATP per turn × about 100 turns per second. The 100 turns per second is an estimate quoted from earlier work, not a direct measurement.","section":"story","topic":"numbers","sources":["fact:A22","fact:A14","stat:Turn rate in mitochondria","ref:watt2010"],"tags":["numbers","speed"],"difficulty":2,"url":"/machines/atp-synthase#story","cites":[{"source":"fact:A22","machine":"atp-synthase","label":"ATP per second, one synthase: 300 per second","section":"story"},{"source":"fact:A14","machine":"atp-synthase","label":"Turn rate in mitochondria: 100 turns per second","section":"story"},{"source":"stat:Turn rate in mitochondria","machine":"atp-synthase","label":"Key number: Turn rate in mitochondria","section":"summary"},{"source":"ref:watt2010","machine":"atp-synthase","label":"Watt et al.","section":"sources","anchor":"ref-watt2010","href":"https://doi.org/10.1073/pnas.1011099107"}]},{"id":"atp-synthase-turbine-breaks","machine":"atp-synthase","kind":"qa","prompt":"Where does the water-turbine analogy for ATP synthase break down?","answer":"Protons do not push on blades: each binds a site on the ring, rides almost a full turn and leaves.","explanation":"Thermal motion turns the ring, and binding and release set the direction. The \"turbine\" also runs backward as a pump.","section":"story","topic":"purpose","sources":["analogy:A water turbine below a dam","stop:machine"],"tags":["analogy","protons"],"difficulty":2,"url":"/machines/atp-synthase#story","cites":[{"source":"analogy:A water turbine below a dam","machine":"atp-synthase","label":"Analogy: A water turbine below a dam","section":"story"},{"source":"stop:machine","machine":"atp-synthase","label":"Big picture: The machine","section":"story"}]},{"id":"atp-synthase-flagellar-link","machine":"atp-synthase","kind":"qa","prompt":"How does the bacterial flagellar motor use a proton gradient differently from ATP synthase?","answer":"It turns the gradient straight into rotation, with no ATP in between.","explanation":"Both are ion-driven rotary motors, and the flagellar export ATPase FliI resembles the F1 α and β subunits.","section":"story","topic":"purpose","sources":["link:flagellar-motor","evolution:Shared parts with the flagellum","ref:imada2007"],"tags":["flagellum","rotation"],"difficulty":2,"url":"/machines/atp-synthase#story","cites":[{"source":"link:flagellar-motor","machine":"atp-synthase","label":"Link to Bacterial flagellar motor","section":"story"},{"source":"evolution:Shared parts with the flagellum","machine":"atp-synthase","label":"Shared parts with the flagellum","section":"summary"},{"source":"ref:imada2007","machine":"atp-synthase","label":"Imada et al.","section":"sources","anchor":"ref-imada2007","href":"https://doi.org/10.1073/pnas.0608090104"}]},{"id":"atp-synthase-first-rotation-method","machine":"atp-synthase","kind":"qa","prompt":"How did Noji and colleagues (1997) first see the γ rotor of F1 (the ATP synthase head) turn?","answer":"They fixed F1 to glass, attached a fluorescent actin filament to γ and filmed it spinning.","explanation":"The system was the α3β3γ part of thermophilic Bacillus PS3 F1, driven by ATP. From the drag on long filaments they set a lower limit of about 40 pN·nm on the torque.","section":"evidence","topic":"numbers","sources":["evidence:f1-rotation-torque","stat:Torque of F1","ref:noji1997"],"tags":["single-molecule","rotation"],"difficulty":2,"url":"/machines/atp-synthase#evidence","cites":[{"source":"evidence:f1-rotation-torque","machine":"atp-synthase","label":"Torque of the F1 motor under high load (Noji H 1997)","section":"evidence","anchor":"ev-f1-rotation-torque"},{"source":"stat:Torque of F1","machine":"atp-synthase","label":"Key number: Torque of F1","section":"summary"},{"source":"ref:noji1997","machine":"atp-synthase","label":"Noji et al.","section":"sources","anchor":"ref-noji1997","href":"https://doi.org/10.1038/386299a0"}]},{"id":"atp-synthase-one-atp-per-step","machine":"atp-synthase","kind":"qa","prompt":"How did Yasuda and colleagues (1998) show that each 120° step of F1 in ATP synthase uses one ATP?","answer":"At low ATP, the rotation rate was about one third of the ATPase rate in solution.","explanation":"The waits between 120° steps also fit one ATP binding per step. So three ATP are used per turn.","section":"evidence","topic":"numbers","sources":["evidence:f1-steps-one-atp","stat:ATP per full turn","ref:yasuda1998"],"tags":["single-molecule","atp"],"difficulty":3,"url":"/machines/atp-synthase#evidence","cites":[{"source":"evidence:f1-steps-one-atp","machine":"atp-synthase","label":"ATP used per 120° step of γ (Yasuda R 1998)","section":"evidence","anchor":"ev-f1-steps-one-atp"},{"source":"stat:ATP per full turn","machine":"atp-synthase","label":"Key number: ATP per full turn","section":"summary"},{"source":"ref:yasuda1998","machine":"atp-synthase","label":"Yasuda et al.","section":"sources","anchor":"ref-yasuda1998","href":"https://doi.org/10.1016/s0092-8674(00)81456-7"}]},{"id":"atp-synthase-work-per-step","machine":"atp-synthase","kind":"qa","prompt":"In F1 of ATP synthase, how does the work done in one 120° step compare with the free energy of one ATP?","answer":"They are about equal: about 90 pN·nm against 80–110 pN·nm.","explanation":"So F1 turns nearly all the energy of an ATP into rotation. The authors note the scatter does not exclude efficiencies near 50%.","section":"evidence","topic":"numbers","sources":["evidence:f1-work-per-step","stat:Work per 120° step","ref:yasuda1998"],"tags":["efficiency","torque"],"difficulty":2,"url":"/machines/atp-synthase#evidence","cites":[{"source":"evidence:f1-work-per-step","machine":"atp-synthase","label":"Mechanical work done in one 120° step (Yasuda R 1998)","section":"evidence","anchor":"ev-f1-work-per-step"},{"source":"stat:Work per 120° step","machine":"atp-synthase","label":"Key number: Work per 120° step","section":"summary"},{"source":"ref:yasuda1998","machine":"atp-synthase","label":"Yasuda et al.","section":"sources","anchor":"ref-yasuda1998","href":"https://doi.org/10.1016/s0092-8674(00)81456-7"}]},{"id":"atp-synthase-hand-cranked-synthesis","machine":"atp-synthase","kind":"qa","prompt":"What did Itoh and colleagues (2004) show by turning γ of isolated F1 with magnets in the synthesis direction?","answer":"ATP appeared, so turning the shaft alone is enough to make ATP.","explanation":"There was no proton gradient, and ATP appeared only when γ turned the right way. In the whole ATP synthase, the protons only supply the turning.","section":"evidence","topic":"numbers","sources":["evidence:f1-mechanical-synthesis","step:ATP forms and leaves","ref:itoh2004"],"tags":["rotation","synthesis"],"difficulty":2,"url":"/machines/atp-synthase#evidence","cites":[{"source":"evidence:f1-mechanical-synthesis","machine":"atp-synthase","label":"ATP made by forcing γ to turn (Itoh H 2004)","section":"evidence","anchor":"ev-f1-mechanical-synthesis"},{"source":"step:ATP forms and leaves","machine":"atp-synthase","label":"Step: ATP forms and leaves","section":"mechanism"},{"source":"ref:itoh2004","machine":"atp-synthase","label":"Itoh et al.","section":"sources","anchor":"ref-itoh2004","href":"https://doi.org/10.1038/nature02212"}]},{"id":"atp-synthase-substeps","machine":"atp-synthase","kind":"cloze","prompt":"Each 120° step of the F1 rotor of ATP synthase splits into substeps of about {{80° and 40°}}.","answer":"80° and 40°","explanation":"They were first reported as about 90° and 30° (gold bead, high-speed camera); a slow β mutant later let the same group refine them to 81° and 39°.","section":"evidence","topic":"numbers","sources":["evidence:f1-substeps-80-40","evidence:f1-substeps-90-30","stat:Step size","ref:shimabukuro2003"],"tags":["substeps","rotation"],"difficulty":2,"url":"/machines/atp-synthase#evidence","cites":[{"source":"evidence:f1-substeps-80-40","machine":"atp-synthase","label":"Refined substep angles (Shimabukuro K 2003)","section":"evidence","anchor":"ev-f1-substeps-80-40"},{"source":"evidence:f1-substeps-90-30","machine":"atp-synthase","label":"Substeps within each 120° step (Yasuda R 2001)","section":"evidence","anchor":"ev-f1-substeps-90-30"},{"source":"stat:Step size","machine":"atp-synthase","label":"Key number: Step size","section":"summary"},{"source":"ref:shimabukuro2003","machine":"atp-synthase","label":"Shimabukuro et al.","section":"sources","anchor":"ref-shimabukuro2003","href":"https://doi.org/10.1073/pnas.2434983100"}]},{"id":"atp-synthase-c8-method","machine":"atp-synthase","kind":"qa","prompt":"How did Watt and colleagues (2010) find that the animal ATP synthase c-ring has 8 subunits?","answer":"X-ray crystallography of the F1–c-ring complex from cow heart mitochondria.","explanation":"Eight protons and three ATP per turn give 2.7 H+/ATP. That is a ratio from structure, not a thermodynamic measurement.","section":"evidence","topic":"numbers","sources":["evidence:c8-ring-bovine","stat:H+ per ATP in animals","ref:watt2010"],"tags":["c-ring","x-ray"],"difficulty":2,"url":"/machines/atp-synthase#evidence","cites":[{"source":"evidence:c8-ring-bovine","machine":"atp-synthase","label":"Number of c subunits in the mammalian c-ring (Watt IN 2010)","section":"evidence","anchor":"ev-c8-ring-bovine"},{"source":"stat:H+ per ATP in animals","machine":"atp-synthase","label":"Key number: H+ per ATP in animals","section":"summary"},{"source":"ref:watt2010","machine":"atp-synthase","label":"Watt et al.","section":"sources","anchor":"ref-watt2010","href":"https://doi.org/10.1073/pnas.1011099107"}]},{"id":"atp-synthase-equilibrium-method","machine":"atp-synthase","kind":"qa","prompt":"How did Petersen and colleagues (2012) measure the H+/ATP ratio of ATP synthase in lipid vesicles?","answer":"For several ATP/(ADP·phosphate) ratios, they found the proton gradient at which the enzyme neither made nor split ATP; the slope gives H+/ATP.","explanation":"This equilibrium method measures the thermodynamic ratio directly, rather than reading it off the c-ring size.","section":"evidence","topic":"numbers","sources":["evidence:h-per-atp-yeast-chloroplast","ref:petersen2012"],"tags":["h-per-atp","method"],"difficulty":3,"url":"/machines/atp-synthase#evidence","cites":[{"source":"evidence:h-per-atp-yeast-chloroplast","machine":"atp-synthase","label":"Thermodynamic H+/ATP ratio, yeast mitochondrial and chloroplast enzymes measured side by side (Petersen J 2012)","section":"evidence","anchor":"ev-h-per-atp-yeast-chloroplast"},{"source":"ref:petersen2012","machine":"atp-synthase","label":"Petersen et al.","section":"sources","anchor":"ref-petersen2012","href":"https://doi.org/10.1073/pnas.1202799109"}]},{"id":"atp-synthase-measured-vs-structural","machine":"atp-synthase","kind":"qa","prompt":"How do the measured H+/ATP ratios of yeast and chloroplast ATP synthase compare with the ratios from their c-ring sizes?","answer":"They are lower: 2.9 against 3.3 (yeast) and 3.9 against 4.7 (chloroplast).","explanation":"Pooled chloroplast data give 4.0 protons per ATP, 85% of the structural 4.7.","section":"evidence","topic":"debate","sources":["evidence:h-per-atp-yeast-chloroplast","evidence:dg-atp-synthesis-standard","mechanism:One turn costs n protons","ref:petersen2012","ref:turina2016"],"tags":["h-per-atp","contested"],"difficulty":2,"url":"/machines/atp-synthase#evidence","cites":[{"source":"evidence:h-per-atp-yeast-chloroplast","machine":"atp-synthase","label":"Thermodynamic H+/ATP ratio, yeast mitochondrial and chloroplast enzymes measured side by side (Petersen J 2012)","section":"evidence","anchor":"ev-h-per-atp-yeast-chloroplast"},{"source":"evidence:dg-atp-synthesis-standard","machine":"atp-synthase","label":"Standard free energy of ATP synthesis and pooled chloroplast H+/ATP (Turina P 2016)","section":"evidence","anchor":"ev-dg-atp-synthesis-standard"},{"source":"mechanism:One turn costs n protons","machine":"atp-synthase","label":"Step: One turn costs n protons","section":"mechanism"},{"source":"ref:petersen2012","machine":"atp-synthase","label":"Petersen et al.","section":"sources","anchor":"ref-petersen2012","href":"https://doi.org/10.1073/pnas.1202799109"},{"source":"ref:turina2016","machine":"atp-synthase","label":"Turina et al.","section":"sources","anchor":"ref-turina2016","href":"https://doi.org/10.1016/j.bbabio.2016.02.019"}]},{"id":"flagellar-motor-what-it-turns","machine":"flagellar-motor","kind":"qa","prompt":"What does the bacterial flagellar motor turn?","answer":"A long helical filament (the flagellum).","explanation":"The motor sits in the cell envelope; the turning filament works as the cell's propeller.","section":"summary","topic":"purpose","sources":["machine:summary","component:FliC (flagellin)"],"tags":["rotation","swimming"],"difficulty":1,"url":"/machines/flagellar-motor#summary","cites":[{"source":"machine:summary","machine":"flagellar-motor","label":"Summary","section":"summary"},{"source":"component:FliC (flagellin)","machine":"flagellar-motor","label":"Part: FliC (flagellin)","section":"summary"}]},{"id":"flagellar-motor-why-reverse","machine":"flagellar-motor","kind":"qa","prompt":"Why does a bacterium need its flagellar motor to reverse?","answer":"Switching between runs and tumbles steers the cell toward food.","explanation":"Counterclockwise rotation gives runs; a short spell of clockwise rotation gives a tumble and a new direction.","section":"summary","topic":"purpose","sources":["machine:summary","story:question"],"tags":["chemotaxis","switch"],"difficulty":1,"url":"/machines/flagellar-motor#summary","cites":[{"source":"machine:summary","machine":"flagellar-motor","label":"Summary","section":"summary"},{"source":"story:question","machine":"flagellar-motor","label":"Big picture","section":"story"}]},{"id":"flagellar-motor-fuel-by-species","machine":"flagellar-motor","kind":"cloze","prompt":"The E. coli flagellar motor runs on an inward flow of protons; the Vibrio polar flagellar motor runs on an inward flow of {{sodium ions}}.","answer":"sodium ions","explanation":"Both ions flow into the cell across the inner membrane. Vibrio uses PomA/PomB stator units instead of MotA/MotB.","section":"summary","topic":"purpose","sources":["machine:energy","species:Vibrio polar motor","component:PomA"],"tags":["ions","energy"],"difficulty":1,"url":"/machines/flagellar-motor#summary","cites":[{"source":"machine:energy","machine":"flagellar-motor","label":"Summary","section":"summary"},{"source":"species:Vibrio polar motor","machine":"flagellar-motor","label":"Vibrio polar motor","section":"summary"},{"source":"component:PomA","machine":"flagellar-motor","label":"Part: PomA","section":"summary"}]},{"id":"flagellar-motor-stator-build","machine":"flagellar-motor","kind":"qa","prompt":"What subunits make one stator unit of the flagellar motor, and how are they arranged?","answer":"A ring of five MotA around two MotB (MotA5B2).","explanation":"Deme and colleagues found the same 5:2 build in stator units from Vibrio, Clostridium and Bacillus.","section":"summary","topic":"parts","sources":["evidence:stator-mota5b2","mechanism:Ions enter the stator","ref:deme2020"],"tags":["stator","structure"],"difficulty":1,"url":"/machines/flagellar-motor#summary","cites":[{"source":"evidence:stator-mota5b2","machine":"flagellar-motor","label":"Subunit stoichiometry of the stator unit (Deme JC 2020)","section":"evidence","anchor":"ev-stator-mota5b2"},{"source":"mechanism:Ions enter the stator","machine":"flagellar-motor","label":"Step: Ions enter the stator","section":"mechanism"},{"source":"ref:deme2020","machine":"flagellar-motor","label":"Deme et al.","section":"sources","anchor":"ref-deme2020","href":"https://doi.org/10.1038/s41564-020-0788-8"}]},{"id":"flagellar-motor-motb-role","machine":"flagellar-motor","kind":"qa","prompt":"In the flagellar motor stator unit, what two jobs does MotB do?","answer":"It anchors the stator unit to the cell wall (peptidoglycan) and carries the aspartate that binds the ion.","explanation":"Because MotB is held fixed by its anchor, the MotA ring can turn around it.","section":"summary","topic":"parts","sources":["component:MotB","mechanism:Ions enter the stator"],"tags":["stator","parts"],"difficulty":2,"url":"/machines/flagellar-motor#summary","cites":[{"source":"component:MotB","machine":"flagellar-motor","label":"Part: MotB","section":"summary"},{"source":"mechanism:Ions enter the stator","machine":"flagellar-motor","label":"Step: Ions enter the stator","section":"mechanism"}]},{"id":"flagellar-motor-chey-site","machine":"flagellar-motor","kind":"cloze","prompt":"Phosphorylated CheY reverses the flagellar motor by binding the N-terminus of {{FliM}} on the C-ring.","answer":"FliM","explanation":"FliM is the middle ring of the C-ring; in the clockwise structure all 34 FliM carry a CheY.","section":"summary","topic":"parts","sources":["component:FliM","step:CheY binds the switch","ref:lee2001"],"tags":["switch","chemotaxis"],"difficulty":1,"url":"/machines/flagellar-motor#summary","cites":[{"source":"component:FliM","machine":"flagellar-motor","label":"Part: FliM","section":"summary"},{"source":"step:CheY binds the switch","machine":"flagellar-motor","label":"Step: CheY binds the switch","section":"mechanism"},{"source":"ref:lee2001","machine":"flagellar-motor","label":"Lee et al.","section":"sources","anchor":"ref-lee2001","href":"https://doi.org/10.1038/83053"}]},{"id":"flagellar-motor-stall-torque","machine":"flagellar-motor","kind":"qa","prompt":"About how much torque does a fully induced E. coli flagellar motor give at high load?","answer":"About 1,260 pN·nm.","explanation":"Measured with 1 µm beads at about 63 Hz, close to stall. One stator unit gives about 146 pN·nm.","section":"summary","topic":"numbers","sources":["stat:Stall torque, E. coli (fully induced)","evidence:stall-torque-fully-induced","ref:reid2006"],"tags":["torque"],"difficulty":2,"url":"/machines/flagellar-motor#summary","cites":[{"source":"stat:Stall torque, E. coli (fully induced)","machine":"flagellar-motor","label":"Key number: Stall torque, E. coli (fully induced)","section":"summary"},{"source":"evidence:stall-torque-fully-induced","machine":"flagellar-motor","label":"Torque of a fully induced E. coli motor at high load (Reid SW 2006)","section":"evidence","anchor":"ev-stall-torque-fully-induced"},{"source":"ref:reid2006","machine":"flagellar-motor","label":"Reid et al.","section":"sources","anchor":"ref-reid2006","href":"https://doi.org/10.1073/pnas.0509932103"}]},{"id":"flagellar-motor-steps-vs-flig","machine":"flagellar-motor","kind":"qa","prompt":"The flagellar rotor takes 26 steps per turn at low driving force. Why can those steps no longer be read as one step per FliG?","answer":"A Salmonella C-ring has 34 FliG subunits, not 26.","explanation":"Sowa and colleagues linked 26 to the FliG ring; later cryo-EM (Tan et al. 2024) counted 34 FliG, so the two counts differ.","section":"summary","topic":"debate","sources":["stat:Steps per turn","evidence:steps-per-revolution","evidence:c-ring-composition","ref:sowa2005","ref:tan2024"],"tags":["steps","c-ring"],"difficulty":2,"url":"/machines/flagellar-motor#summary","cites":[{"source":"stat:Steps per turn","machine":"flagellar-motor","label":"Key number: Steps per turn","section":"summary"},{"source":"evidence:steps-per-revolution","machine":"flagellar-motor","label":"Steps per revolution of the rotor (Sowa Y 2005)","section":"evidence","anchor":"ev-steps-per-revolution"},{"source":"evidence:c-ring-composition","machine":"flagellar-motor","label":"Subunit counts and size of the C ring-containing motor (Tan J 2024)","section":"evidence","anchor":"ev-c-ring-composition"},{"source":"ref:sowa2005","machine":"flagellar-motor","label":"Sowa et al.","section":"sources","anchor":"ref-sowa2005","href":"https://doi.org/10.1038/nature04003"},{"source":"ref:tan2024","machine":"flagellar-motor","label":"Tan et al.","section":"sources","anchor":"ref-tan2024","href":"https://doi.org/10.1038/s41422-024-01017-z"}]},{"id":"flagellar-motor-wider-ring-proposal","machine":"flagellar-motor","kind":"qa","prompt":"What proposed (not yet done) way could build a stronger flagellar motor, copying how nature raises torque?","answer":"Transplant scaffold rings that hold more stator units at a wider radius.","explanation":"High-torque motors such as those of Vibrio and Campylobacter use scaffolds to place more stator units further out. The idea's status is \"proposed\".","section":"summary","topic":"debate","sources":["frontier:Torque from stator-unit placement","evolution:Wider rings for more torque","ref:beeby2016"],"tags":["torque","frontier"],"difficulty":2,"url":"/machines/flagellar-motor#summary","cites":[{"source":"frontier:Torque from stator-unit placement","machine":"flagellar-motor","label":"Open question: Torque from stator-unit placement","section":"summary"},{"source":"evolution:Wider rings for more torque","machine":"flagellar-motor","label":"Wider rings for more torque","section":"summary"},{"source":"ref:beeby2016","machine":"flagellar-motor","label":"Beeby et al.","section":"sources","anchor":"ref-beeby2016","href":"https://doi.org/10.1073/pnas.1518952113"}]},{"id":"flagellar-motor-proton-site","machine":"flagellar-motor","kind":"qa","prompt":"In the E. coli flagellar stator unit, which residue takes up the incoming proton?","answer":"MotB Asp32 (a conserved aspartate on MotB).","explanation":"The proton enters a channel between MotA and MotB; Asp32 sits inside a ring of five MotA Thr209.","section":"mechanism","topic":"cycle","sources":["step:Ions flow through the stators","mechanism:Ions enter the stator","ref:santiveri2020"],"tags":["stator","ions"],"difficulty":2,"url":"/machines/flagellar-motor#mechanism","cites":[{"source":"step:Ions flow through the stators","machine":"flagellar-motor","label":"Step: Ions flow through the stators","section":"mechanism"},{"source":"mechanism:Ions enter the stator","machine":"flagellar-motor","label":"Step: Ions enter the stator","section":"mechanism"},{"source":"ref:santiveri2020","machine":"flagellar-motor","label":"Santiveri et al.","section":"sources","anchor":"ref-santiveri2020","href":"https://doi.org/10.1016/j.cell.2020.08.016"}]},{"id":"flagellar-motor-36-degrees","machine":"flagellar-motor","kind":"cloze","prompt":"In the proposed model of the flagellar stator, each ion turns the MotA ring about 36°, so {{ten}} ions turn MotA once.","answer":"ten","explanation":"This is a model built from the lopsided 5:2 structures; the MotA step has not been seen directly.","section":"mechanism","topic":"cycle","sources":["step:MotA turns around MotB","evidence:stator-36-degree-model","ref:deme2020"],"tags":["stator","rotation"],"difficulty":2,"url":"/machines/flagellar-motor#mechanism","cites":[{"source":"step:MotA turns around MotB","machine":"flagellar-motor","label":"Step: MotA turns around MotB","section":"mechanism"},{"source":"evidence:stator-36-degree-model","machine":"flagellar-motor","label":"Proposed rotation of the MotA ring per ion (Deme JC 2020)","section":"evidence","anchor":"ev-stator-36-degree-model"},{"source":"ref:deme2020","machine":"flagellar-motor","label":"Deme et al.","section":"sources","anchor":"ref-deme2020","href":"https://doi.org/10.1038/s41564-020-0788-8"}]},{"id":"flagellar-motor-ratchet-direction","machine":"flagellar-motor","kind":"qa","prompt":"In the Deme model of the flagellar stator, why do ions always push the MotA ring the same way?","answer":"The 5:2 ring is lopsided, so the two MotB aspartates sit in different surroundings; binding at one while the other lets go acts like a ratchet.","explanation":"Ions bind and leave the two MotB helices in turn, and MotB stays fixed by its cell-wall anchor.","section":"mechanism","topic":"cycle","sources":["step:MotA turns around MotB","ref:deme2020"],"tags":["stator","ratchet"],"difficulty":3,"url":"/machines/flagellar-motor#mechanism","cites":[{"source":"step:MotA turns around MotB","machine":"flagellar-motor","label":"Step: MotA turns around MotB","section":"mechanism"},{"source":"ref:deme2020","machine":"flagellar-motor","label":"Deme et al.","section":"sources","anchor":"ref-deme2020","href":"https://doi.org/10.1038/s41564-020-0788-8"}]},{"id":"flagellar-motor-torque-per-unit","machine":"flagellar-motor","kind":"qa","prompt":"Torque is force times lever. What force and lever radius give each enteric flagellar stator unit its torque of about 146 pN·nm?","answer":"About 7 pN at about 20 nm from the axis.","explanation":"The 7.3 pN is derived (146 pN·nm ÷ 20 nm), not measured directly. A full set of units gives the 1,260 pN·nm of an E. coli motor.","section":"mechanism","topic":"cycle","sources":["step:MotA drives FliG like a gear","evidence:force-per-stator-model","ref:beeby2016"],"tags":["torque","lever"],"difficulty":2,"url":"/machines/flagellar-motor#mechanism","cites":[{"source":"step:MotA drives FliG like a gear","machine":"flagellar-motor","label":"Step: MotA drives FliG like a gear","section":"mechanism"},{"source":"evidence:force-per-stator-model","machine":"flagellar-motor","label":"Force per stator unit and predicted motor torques (Beeby M 2016)","section":"evidence","anchor":"ev-force-per-stator-model"},{"source":"ref:beeby2016","machine":"flagellar-motor","label":"Beeby et al.","section":"sources","anchor":"ref-beeby2016","href":"https://doi.org/10.1073/pnas.1518952113"}]},{"id":"flagellar-motor-drive-train","machine":"flagellar-motor","kind":"qa","prompt":"In the flagellar motor, what part does the rod spin inside as it passes through the cell wall and outer membrane?","answer":"The LP-ring, a bushing that does not turn.","explanation":"Torque goes C-ring → MS-ring → rod → hook → filament; the LP-ring (FlgH, FlgI) is anchored and lets the rod spin.","section":"mechanism","topic":"cycle","sources":["step:The rotor turns counterclockwise","mechanism:Torque travels up the drive shaft","ref:johnson2021"],"tags":["rotor","bearing"],"difficulty":2,"url":"/machines/flagellar-motor#mechanism","cites":[{"source":"step:The rotor turns counterclockwise","machine":"flagellar-motor","label":"Step: The rotor turns counterclockwise","section":"mechanism"},{"source":"mechanism:Torque travels up the drive shaft","machine":"flagellar-motor","label":"Step: Torque travels up the drive shaft","section":"mechanism"},{"source":"ref:johnson2021","machine":"flagellar-motor","label":"Johnson et al.","section":"sources","anchor":"ref-johnson2021","href":"https://doi.org/10.1038/s41564-021-00895-y"}]},{"id":"flagellar-motor-switch-flig-flip","machine":"flagellar-motor","kind":"qa","prompt":"When CheY-P binds the flagellar C-ring, how do the FliG domains move, and what does that change for the stator units?","answer":"They turn by 180°, so the stator units meet FliG from the inside of the ring instead of the outside.","explanation":"The ring keeps its 34-fold symmetry; only the contact side changes.","section":"mechanism","topic":"cycle","sources":["mechanism:CheY-P flips the switch","evidence:switch-fligc-180-johnson","ref:johnson2024","ref:tan2024"],"tags":["switch","c-ring"],"difficulty":2,"url":"/machines/flagellar-motor#mechanism","cites":[{"source":"mechanism:CheY-P flips the switch","machine":"flagellar-motor","label":"Step: CheY-P flips the switch","section":"mechanism"},{"source":"evidence:switch-fligc-180-johnson","machine":"flagellar-motor","label":"FliG movement and stator position in the two rotation states (Johnson S 2024)","section":"evidence","anchor":"ev-switch-fligc-180-johnson"},{"source":"ref:johnson2024","machine":"flagellar-motor","label":"Johnson et al.","section":"sources","anchor":"ref-johnson2024","href":"https://doi.org/10.1038/s41564-024-01630-z"},{"source":"ref:tan2024","machine":"flagellar-motor","label":"Tan et al.","section":"sources","anchor":"ref-tan2024","href":"https://doi.org/10.1038/s41422-024-01017-z"}]},{"id":"flagellar-motor-gear-reversal","machine":"flagellar-motor","kind":"qa","prompt":"MotA keeps turning clockwise in both states of the flagellar motor. Why does moving its contact from the outer to the inner face of the FliG ring reverse the rotor?","answer":"Gears that touch on their outer faces turn opposite ways; a gear driving the inside of a ring turns it the same way.","explanation":"So the rotor turns counterclockwise with outside contact and clockwise with inside contact, without reversing the ion flow.","section":"mechanism","topic":"cycle","sources":["step:The rotor turns clockwise","mechanism:CheY-P flips the switch","ref:johnson2024"],"tags":["switch","gear"],"difficulty":3,"url":"/machines/flagellar-motor#mechanism","cites":[{"source":"step:The rotor turns clockwise","machine":"flagellar-motor","label":"Step: The rotor turns clockwise","section":"mechanism"},{"source":"mechanism:CheY-P flips the switch","machine":"flagellar-motor","label":"Step: CheY-P flips the switch","section":"mechanism"},{"source":"ref:johnson2024","machine":"flagellar-motor","label":"Johnson et al.","section":"sources","anchor":"ref-johnson2024","href":"https://doi.org/10.1038/s41564-024-01630-z"}]},{"id":"flagellar-motor-catch-bond","machine":"flagellar-motor","kind":"qa","prompt":"Why do more stator units bind the flagellar motor when the load rises?","answer":"The MotB anchor forms a catch bond that holds longer under force.","explanation":"Stator units bind and leave all the time; under high load each bound unit stays longer, so more gather and torque rises.","section":"mechanism","topic":"cycle","sources":["mechanism:Load sets the number of stator units","evidence:stator-catch-bond","ref:nord2017"],"tags":["stator","load"],"difficulty":2,"url":"/machines/flagellar-motor#mechanism","cites":[{"source":"mechanism:Load sets the number of stator units","machine":"flagellar-motor","label":"Step: Load sets the number of stator units","section":"mechanism"},{"source":"evidence:stator-catch-bond","machine":"flagellar-motor","label":"Stator lifetime against applied force (Nord AL 2017)","section":"evidence","anchor":"ev-stator-catch-bond"},{"source":"ref:nord2017","machine":"flagellar-motor","label":"Nord et al.","section":"sources","anchor":"ref-nord2017","href":"https://doi.org/10.1073/pnas.1716002114"}]},{"id":"flagellar-motor-coast-distance","machine":"flagellar-motor","kind":"qa","prompt":"About how far does a swimming bacterium coast after its flagellar motors stop?","answer":"About 0.1 Å, less than an atom's width.","explanation":"At this size water feels like thick syrup (Reynolds number about 3 × 10^-5), so drag stops the cell almost at once. Purcell's order-of-magnitude estimate.","section":"story","topic":"numbers","sources":["fact:F6","stop:cell","ref:purcell1977"],"tags":["low-reynolds","swimming"],"difficulty":2,"url":"/machines/flagellar-motor#story","cites":[{"source":"fact:F6","machine":"flagellar-motor","label":"How far the cell coasts when its motors stop: 0.1 Å","section":"story"},{"source":"stop:cell","machine":"flagellar-motor","label":"Big picture: One cell","section":"story"},{"source":"ref:purcell1977","machine":"flagellar-motor","label":"Purcell 1977","section":"sources","href":"https://doi.org/10.1119/1.10903"}]},{"id":"flagellar-motor-reverse-gear-analogy","machine":"flagellar-motor","kind":"qa","prompt":"The flagellar motor is like a car with a reverse gear. Where does that analogy break?","answer":"Reversing does not drive the cell backward; it breaks up the bundle and the cell tumbles to a new heading.","section":"story","topic":"purpose","sources":["analogy:A car with a reverse gear"],"tags":["analogy","switch"],"difficulty":2,"url":"/machines/flagellar-motor#story","cites":[{"source":"analogy:A car with a reverse gear","machine":"flagellar-motor","label":"Analogy: A car with a reverse gear","section":"story"}]},{"id":"flagellar-motor-protons-per-turn","machine":"flagellar-motor","kind":"qa","prompt":"About how many protons pass through the flagellar motor of Streptococcus per rotor turn?","answer":"About 1,240 (± 240).","explanation":"Measured in Streptococcus, not E. coli, so it should not be multiplied by E. coli motor speeds. Proton flux rises in direct proportion to speed.","section":"story","topic":"numbers","sources":["fact:F10","evidence:proton-flux-speed","ref:bnid109759"],"tags":["protons","energy"],"difficulty":2,"url":"/machines/flagellar-motor#story","cites":[{"source":"fact:F10","machine":"flagellar-motor","label":"Protons that pass per rotor turn: 1,240 protons per turn","section":"story"},{"source":"evidence:proton-flux-speed","machine":"flagellar-motor","label":"Proton flux through the motor against speed (Meister M 1987)","section":"evidence","anchor":"ev-proton-flux-speed"},{"source":"ref:bnid109759","machine":"flagellar-motor","label":"BNID 109759","section":"sources","href":"https://bionumbers.hms.harvard.edu/bionumber.aspx?id=109759"}]},{"id":"flagellar-motor-atp-synthase-link","machine":"flagellar-motor","kind":"qa","prompt":"Which part of the flagellar motor resembles the F1 α and β subunits of ATP synthase?","answer":"The export ATPase FliI.","explanation":"FliJ also looks like the γ coiled-coil, so the flagellar export machine and the rotary ATPases likely share an ancestor.","section":"story","topic":"parts","sources":["link:atp-synthase","evolution:Shared parts with ATP synthase","ref:imada2007","ref:ibuki2011"],"tags":["evolution","atp-synthase"],"difficulty":2,"url":"/machines/flagellar-motor#story","cites":[{"source":"link:atp-synthase","machine":"flagellar-motor","label":"Link to ATP synthase","section":"story"},{"source":"evolution:Shared parts with ATP synthase","machine":"flagellar-motor","label":"Shared parts with ATP synthase","section":"summary"},{"source":"ref:imada2007","machine":"flagellar-motor","label":"Imada et al.","section":"sources","anchor":"ref-imada2007","href":"https://doi.org/10.1073/pnas.0608090104"},{"source":"ref:ibuki2011","machine":"flagellar-motor","label":"Ibuki et al.","section":"sources","anchor":"ref-ibuki2011","href":"https://doi.org/10.1038/nsmb.1977"}]},{"id":"flagellar-motor-how-torque-measured","machine":"flagellar-motor","kind":"qa","prompt":"How did Reid and colleagues measure the torque of single E. coli flagellar motors?","answer":"They tracked a 1 µm bead on a flagellar stub by laser interferometry and took torque = speed × bead drag coefficient.","explanation":"At these low speeds torque sits near its stall value, so a fully induced motor gives the motor's maximum, about 1,260 pN·nm.","section":"evidence","topic":"numbers","sources":["evidence:stall-torque-fully-induced","ref:reid2006"],"tags":["bead-assay","torque"],"difficulty":2,"url":"/machines/flagellar-motor#evidence","cites":[{"source":"evidence:stall-torque-fully-induced","machine":"flagellar-motor","label":"Torque of a fully induced E. coli motor at high load (Reid SW 2006)","section":"evidence","anchor":"ev-stall-torque-fully-induced"},{"source":"ref:reid2006","machine":"flagellar-motor","label":"Reid et al.","section":"sources","anchor":"ref-reid2006","href":"https://doi.org/10.1073/pnas.0509932103"}]},{"id":"flagellar-motor-resurrection-count","machine":"flagellar-motor","kind":"qa","prompt":"In \"resurrection\" experiments on paralysed E. coli flagellar motors, how was the number of stator units counted?","answer":"By counting the discrete jumps in bead speed as new stator units joined the motor.","explanation":"Reid and colleagues counted at least 11 levels, beating the older estimate of eight; the jumps got smaller at high numbers.","section":"evidence","topic":"numbers","sources":["evidence:stator-number-resurrection","evidence:resurrection-eight-steps","ref:reid2006","ref:blair1988"],"tags":["stator","resurrection"],"difficulty":2,"url":"/machines/flagellar-motor#evidence","cites":[{"source":"evidence:stator-number-resurrection","machine":"flagellar-motor","label":"Maximum number of torque-generating units (Reid SW 2006)","section":"evidence","anchor":"ev-stator-number-resurrection"},{"source":"evidence:resurrection-eight-steps","machine":"flagellar-motor","label":"Torque restoration in repaired motors (Blair DF 1988)","section":"evidence","anchor":"ev-resurrection-eight-steps"},{"source":"ref:reid2006","machine":"flagellar-motor","label":"Reid et al.","section":"sources","anchor":"ref-reid2006","href":"https://doi.org/10.1073/pnas.0509932103"},{"source":"ref:blair1988","machine":"flagellar-motor","label":"Blair & Berg, Science 1988","section":"sources","anchor":"ref-blair1988","href":"https://doi.org/10.1126/science.2849208"}]},{"id":"flagellar-motor-zero-load-one-unit","machine":"flagellar-motor","kind":"qa","prompt":"Near zero load, how does the speed of the E. coli flagellar motor depend on the number of stator units?","answer":"It does not: one unit gives the same speed as many (about 300 Hz at 23 °C).","explanation":"Yuan and Berg used 60 nm gold spheres on hooks with no filament; a sphere jumped from rest to full speed when the first unit arrived.","section":"evidence","topic":"numbers","sources":["evidence:zero-load-one-stator","ref:yuan2008"],"tags":["speed","load"],"difficulty":3,"url":"/machines/flagellar-motor#evidence","cites":[{"source":"evidence:zero-load-one-stator","machine":"flagellar-motor","label":"Speed near zero load against number of stator units (Yuan J 2008)","section":"evidence","anchor":"ev-zero-load-one-stator"},{"source":"ref:yuan2008","machine":"flagellar-motor","label":"Yuan & Berg, PNAS 2008","section":"sources","anchor":"ref-yuan2008","href":"https://doi.org/10.1073/pnas.0711539105"}]},{"id":"flagellar-motor-torque-speed-knee","machine":"flagellar-motor","kind":"cloze","prompt":"At 23 °C the torque of the E. coli flagellar motor stays roughly constant up to a knee near {{200 Hz}}, then falls to zero near 350 Hz.","answer":"200 Hz","explanation":"Chen and Berg varied the load with Ficoll. Later work puts the knee at 170–175 Hz.","section":"evidence","topic":"numbers","sources":["evidence:knee-speed","stat:Knee speed, E. coli (23 °C)","evidence:zero-torque-speed","ref:chen2000"],"tags":["torque-speed"],"difficulty":2,"url":"/machines/flagellar-motor#evidence","cites":[{"source":"evidence:knee-speed","machine":"flagellar-motor","label":"Knee of the torque–speed curve (Chen X 2000)","section":"evidence","anchor":"ev-knee-speed"},{"source":"stat:Knee speed, E. coli (23 °C)","machine":"flagellar-motor","label":"Key number: Knee speed, E. coli (23 °C)","section":"summary"},{"source":"evidence:zero-torque-speed","machine":"flagellar-motor","label":"Speed at which torque reaches zero (Chen X 2000)","section":"evidence","anchor":"ev-zero-torque-speed"},{"source":"ref:chen2000","machine":"flagellar-motor","label":"Chen & Berg, Biophys J 2000","section":"sources","anchor":"ref-chen2000","href":"https://doi.org/10.1016/s0006-3495(00)76662-8"}]},{"id":"flagellar-motor-backward-steps","machine":"flagellar-motor","kind":"qa","prompt":"What did the occasional backward steps of the slowed flagellar motor (Sowa et al. 2005) show about each step?","answer":"Each step uses little energy, close to that of one ion crossing.","explanation":"They slowed a Na+-driven chimeric motor in E. coli with low sodium-motive force and few stator units to see 26 steps per turn.","section":"evidence","topic":"cycle","sources":["evidence:steps-per-revolution","mechanism:The motor moves in steps","ref:sowa2005"],"tags":["steps","energy"],"difficulty":3,"url":"/machines/flagellar-motor#evidence","cites":[{"source":"evidence:steps-per-revolution","machine":"flagellar-motor","label":"Steps per revolution of the rotor (Sowa Y 2005)","section":"evidence","anchor":"ev-steps-per-revolution"},{"source":"mechanism:The motor moves in steps","machine":"flagellar-motor","label":"Step: The motor moves in steps","section":"mechanism"},{"source":"ref:sowa2005","machine":"flagellar-motor","label":"Sowa et al.","section":"sources","anchor":"ref-sowa2005","href":"https://doi.org/10.1038/nature04003"}]},{"id":"flagellar-motor-hill-disagreement","machine":"flagellar-motor","kind":"qa","prompt":"Cluzel et al. found a Hill coefficient of about 10 for the flagellar switch; Yuan and Berg found 16.5–20.7. Why the difference?","answer":"Yuan and Berg changed CheY-P faster than motors could change their FliM content; the earlier curve pooled motors that had adapted their FliM.","explanation":"With a fixed make-up the switch is about twice as steep.","section":"evidence","topic":"debate","sources":["evidence:switch-hill-fixed-flim","evidence:switch-ultrasensitivity","stat:Switch sensitivity to CheY-P","ref:yuan2013","ref:cluzel2000"],"tags":["switch","ultrasensitivity"],"difficulty":3,"url":"/machines/flagellar-motor#evidence","cites":[{"source":"evidence:switch-hill-fixed-flim","machine":"flagellar-motor","label":"Hill coefficient of the switch for motors with fixed FliM content (Yuan J 2013)","section":"evidence","anchor":"ev-switch-hill-fixed-flim"},{"source":"evidence:switch-ultrasensitivity","machine":"flagellar-motor","label":"Steepness of the CW bias response to CheY-P (Cluzel P 2000)","section":"evidence","anchor":"ev-switch-ultrasensitivity"},{"source":"stat:Switch sensitivity to CheY-P","machine":"flagellar-motor","label":"Key number: Switch sensitivity to CheY-P","section":"summary"},{"source":"ref:yuan2013","machine":"flagellar-motor","label":"Yuan & Berg, J Mol Biol 2013","section":"sources","anchor":"ref-yuan2013","href":"https://doi.org/10.1016/j.jmb.2013.02.016"},{"source":"ref:cluzel2000","machine":"flagellar-motor","label":"Cluzel et al.","section":"sources","anchor":"ref-cluzel2000","href":"https://doi.org/10.1126/science.287.5458.1652"}]},{"id":"kinesin-direction","machine":"kinesin","kind":"qa","prompt":"Which way along a microtubule does kinesin-1 carry its cargo?","answer":"Toward the plus end.","explanation":"Its two heads take turns: the rear head swings past the bound head and lands 16 nm ahead.","section":"summary","topic":"purpose","sources":["machine:summary","machine:tagline"],"tags":["direction","transport"],"difficulty":1,"url":"/machines/kinesin#summary","cites":[{"source":"machine:summary","machine":"kinesin","label":"Summary","section":"summary"},{"source":"machine:tagline","machine":"kinesin","label":"Summary","section":"summary"}]},{"id":"kinesin-neck-linker-role","machine":"kinesin","kind":"qa","prompt":"Which part of kinesin-1 docks onto the head when ATP binds and pulls the partner head forward?","answer":"The neck linker.","explanation":"It is a short segment of about 13-15 residues after helix α6; papers draw its boundaries differently.","section":"summary","topic":"parts","sources":["component:neck linker","stat:Neck linker","ref:rice1999"],"tags":["neck-linker","parts"],"difficulty":1,"url":"/machines/kinesin#summary","cites":[{"source":"component:neck linker","machine":"kinesin","label":"Part: neck linker","section":"summary"},{"source":"stat:Neck linker","machine":"kinesin","label":"Key number: Neck linker","section":"summary"},{"source":"ref:rice1999","machine":"kinesin","label":"Rice et al.","section":"sources","anchor":"ref-rice1999","href":"https://doi.org/10.1038/45483"}]},{"id":"kinesin-direction-from-neck","machine":"kinesin","kind":"qa","prompt":"Plus-end and minus-end kinesins have nearly identical cores. Which part of a kinesin sets the direction it walks?","answer":"The neck next to the core.","explanation":"Swapping necks or domain order reverses motion, so the core alone does not set direction.","section":"summary","topic":"parts","sources":["evolution:Direction from the neck","ref:endow1998","ref:case1997"],"tags":["direction","evolution"],"difficulty":2,"url":"/machines/kinesin#summary","cites":[{"source":"evolution:Direction from the neck","machine":"kinesin","label":"Direction from the neck","section":"summary"},{"source":"ref:endow1998","machine":"kinesin","label":"Endow et al.","section":"sources","anchor":"ref-endow1998","href":"https://doi.org/10.1126/science.281.5380.1200"},{"source":"ref:case1997","machine":"kinesin","label":"Case et al.","section":"sources","anchor":"ref-case1997","href":"https://doi.org/10.1016/s0092-8674(00)80360-8"}]},{"id":"kinesin-step-size","machine":"kinesin","kind":"cloze","prompt":"Kinesin-1's centre of mass moves {{8 nm}} per step, the length of one tubulin dimer.","answer":"8 nm","explanation":"Each head moves about twice as far (17.3 ± 3.3 nm), because the heads pass each other.","section":"summary","topic":"numbers","sources":["stat:Step size","stat:Head step","ref:svoboda1993"],"tags":["step","numbers"],"difficulty":1,"url":"/machines/kinesin#summary","cites":[{"source":"stat:Step size","machine":"kinesin","label":"Key number: Step size","section":"summary"},{"source":"stat:Head step","machine":"kinesin","label":"Key number: Head step","section":"summary"},{"source":"ref:svoboda1993","machine":"kinesin","label":"Svoboda et al.","section":"sources","anchor":"ref-svoboda1993","href":"https://doi.org/10.1038/365721a0"}]},{"id":"kinesin-steps-per-run","machine":"kinesin","kind":"qa","prompt":"About how many steps does a single kinesin-1 take before it lets go of the microtubule?","answer":"About 100 (a run of about 1 µm).","explanation":"The fitted run length at zero load is 1.12 µm, which is more than 100 steps per encounter with the microtubule.","section":"summary","topic":"numbers","sources":["stat:Run length","machine:summary","ref:milic2014"],"tags":["processivity","numbers"],"difficulty":2,"url":"/machines/kinesin#summary","cites":[{"source":"stat:Run length","machine":"kinesin","label":"Key number: Run length","section":"summary"},{"source":"machine:summary","machine":"kinesin","label":"Summary","section":"summary"},{"source":"ref:milic2014","machine":"kinesin","label":"Milic et al.","section":"sources","anchor":"ref-milic2014","href":"https://doi.org/10.1073/pnas.1410943111"}]},{"id":"kinesin-turnover-time","machine":"kinesin","kind":"cloze","prompt":"With no load, kinesin-1 walks at about 800 nm/s, so one turnover takes about {{10 ms}}.","answer":"10 ms","explanation":"At 8 nm per step, 800 nm/s is about 100 steps per second.","section":"summary","topic":"numbers","sources":["stat:Velocity, no load","fact:s-speed","ref:verbrugge2007"],"tags":["speed","numbers"],"difficulty":2,"url":"/machines/kinesin#summary","cites":[{"source":"stat:Velocity, no load","machine":"kinesin","label":"Key number: Velocity, no load","section":"summary"},{"source":"fact:s-speed","machine":"kinesin","label":"Walking speed with no load: 800 nm/s","section":"story"},{"source":"ref:verbrugge2007","machine":"kinesin","label":"Verbrugge et al.","section":"sources","anchor":"ref-verbrugge2007","href":"https://doi.org/10.1529/biophysj.106.093575"}]},{"id":"kinesin-molecular-shuttle","machine":"kinesin","kind":"qa","prompt":"In a kinesin 'molecular shuttle', what does the kinesin do?","answer":"It is fixed on a surface and pushes microtubules that carry cargo.","explanation":"Surface patterns steer the microtubules, and the ATP supply sets their speed. This use is demonstrated.","section":"summary","topic":"debate","sources":["frontier:Molecular shuttles","ref:hess2001"],"tags":["frontier","engineering"],"difficulty":2,"url":"/machines/kinesin#summary","cites":[{"source":"frontier:Molecular shuttles","machine":"kinesin","label":"Open question: Molecular shuttles","section":"summary"},{"source":"ref:hess2001","machine":"kinesin","label":"Hess et al.","section":"sources","anchor":"ref-hess2001","href":"https://doi.org/10.1016/s1389-0352(01)00029-0"}]},{"id":"kinesin-designed-motor","machine":"kinesin","kind":"qa","prompt":"Unlike kinesin, how does a designed protein motor (a protease-coated hub) move across a peptide lawn?","answer":"By cutting the path behind it.","explanation":"It reaches up to 80 nm/s. Designed protein motors are now possible at lab scale, but they are simpler than kinesin.","section":"summary","topic":"debate","sources":["frontier:Designed protein motors","ref:korosec2024"],"tags":["frontier","design"],"difficulty":2,"url":"/machines/kinesin#summary","cites":[{"source":"frontier:Designed protein motors","machine":"kinesin","label":"Open question: Designed protein motors","section":"summary"},{"source":"ref:korosec2024","machine":"kinesin","label":"Korosec et al.","section":"sources","anchor":"ref-korosec2024","href":"https://doi.org/10.1038/s41467-024-45570-y"}]},{"id":"kinesin-atp-docks-linker","machine":"kinesin","kind":"cloze","prompt":"In kinesin-1, {{ATP binding}} to the bound head makes its neck linker zip onto the head.","answer":"ATP binding","explanation":"This docking is the power stroke: the neck linker ends up pointing to the plus end.","section":"mechanism","topic":"cycle","sources":["mechanism:ATP docks the neck linker","step:ATP binds and the neck linker zips","ref:rice1999"],"tags":["neck-linker","atp","power-stroke"],"difficulty":1,"url":"/machines/kinesin#mechanism","cites":[{"source":"mechanism:ATP docks the neck linker","machine":"kinesin","label":"Step: ATP docks the neck linker","section":"mechanism"},{"source":"step:ATP binds and the neck linker zips","machine":"kinesin","label":"Step: ATP binds and the neck linker zips","section":"mechanism"},{"source":"ref:rice1999","machine":"kinesin","label":"Rice et al.","section":"sources","anchor":"ref-rice1999","href":"https://doi.org/10.1038/45483"}]},{"id":"kinesin-why-docking-needs-atp","machine":"kinesin","kind":"qa","prompt":"Why do ATP binding and neck-linker docking go together in a kinesin-1 head?","answer":"Only the closed, ATP-bound head offers the groove the neck linker docks into.","explanation":"Closing the nucleotide pocket turns two parts of the head (by about 22° and 11°), and that opens a groove along the head.","section":"mechanism","topic":"cycle","sources":["step:ATP binds and the neck linker zips"],"tags":["neck-linker","atp"],"difficulty":3,"url":"/machines/kinesin#mechanism","cites":[{"source":"step:ATP binds and the neck linker zips","machine":"kinesin","label":"Step: ATP binds and the neck linker zips","section":"mechanism"}]},{"id":"kinesin-diffusion-search","machine":"kinesin","kind":"cloze","prompt":"In a kinesin-1 step, {{diffusion}} carries the free head forward; neck-linker docking only sets the direction.","answer":"diffusion","explanation":"The docked neck linker of the bound head holds the tethered head near the forward site, and it lands about 16 nm ahead.","section":"mechanism","topic":"cycle","sources":["step:The free head searches","mechanism:The rear head swings forward","ref:yildiz2004"],"tags":["diffusion","stepping"],"difficulty":2,"url":"/machines/kinesin#mechanism","cites":[{"source":"step:The free head searches","machine":"kinesin","label":"Step: The free head searches","section":"mechanism"},{"source":"mechanism:The rear head swings forward","machine":"kinesin","label":"Step: The rear head swings forward","section":"mechanism"},{"source":"ref:yildiz2004","machine":"kinesin","label":"Yildiz et al.","section":"sources","anchor":"ref-yildiz2004","href":"https://doi.org/10.1126/science.1093753"}]},{"id":"kinesin-adp-release-trigger","machine":"kinesin","kind":"qa","prompt":"What makes a kinesin-1 head release its ADP?","answer":"Binding the microtubule, which opens the nucleotide cleft.","explanation":"A dimer that lands on a microtubule releases only one of its two ADPs, so one head binds the track and the other stays free.","section":"mechanism","topic":"cycle","sources":["mechanism:One head holds the track","step:The front head waits for ATP","ref:shang2014"],"tags":["adp","microtubule"],"difficulty":2,"url":"/machines/kinesin#mechanism","cites":[{"source":"mechanism:One head holds the track","machine":"kinesin","label":"Step: One head holds the track","section":"mechanism"},{"source":"step:The front head waits for ATP","machine":"kinesin","label":"Step: The front head waits for ATP","section":"mechanism"},{"source":"ref:shang2014","machine":"kinesin","label":"Shang et al.","section":"sources","anchor":"ref-shang2014","href":"https://doi.org/10.7554/eLife.04686"}]},{"id":"kinesin-adp-weak-binding","machine":"kinesin","kind":"cloze","prompt":"A kinesin-1 head holding {{ADP}} binds the track only weakly, so it can search or let go.","answer":"ADP","explanation":"A head with an empty pocket grips the track tightly, so the new front head anchors the motor before the rear head lets go.","section":"mechanism","topic":"cycle","sources":["step:The rear head lets go","step:The free head searches","step:It lands and releases ADP"],"tags":["adp","gating"],"difficulty":2,"url":"/machines/kinesin#mechanism","cites":[{"source":"step:The rear head lets go","machine":"kinesin","label":"Step: The rear head lets go","section":"mechanism"},{"source":"step:The free head searches","machine":"kinesin","label":"Step: The free head searches","section":"mechanism"},{"source":"step:It lands and releases ADP","machine":"kinesin","label":"Step: It lands and releases ADP","section":"mechanism"}]},{"id":"kinesin-phosphate-gate","machine":"kinesin","kind":"qa","prompt":"Why does kinesin-1 stay on the track for about 100 steps instead of falling off after one?","answer":"The rear head lets go only after phosphate leaves, and by then the front head is bound.","explanation":"This order keeps one head on the track at all times.","section":"mechanism","topic":"cycle","sources":["step:The rear head lets go","ref:milic2014"],"tags":["processivity","gating","phosphate"],"difficulty":3,"url":"/machines/kinesin#mechanism","cites":[{"source":"step:The rear head lets go","machine":"kinesin","label":"Step: The rear head lets go","section":"mechanism"},{"source":"ref:milic2014","machine":"kinesin","label":"Milic et al.","section":"sources","anchor":"ref-milic2014","href":"https://doi.org/10.1073/pnas.1410943111"}]},{"id":"kinesin-hydrolysis-timing-debate","machine":"kinesin","kind":"qa","prompt":"When kinesin-1 splits its ATP is still argued. What does tracking single heads at 1,000 frames per second suggest?","answer":"The bound head must split its ATP before the free head can land.","explanation":"The animation in the notes shows the split after the front head lands instead.","section":"mechanism","topic":"debate","sources":["step:The rear head hydrolyses ATP","ref:mickolajczyk2015"],"tags":["hydrolysis","debate"],"difficulty":3,"url":"/machines/kinesin#mechanism","cites":[{"source":"step:The rear head hydrolyses ATP","machine":"kinesin","label":"Step: The rear head hydrolyses ATP","section":"mechanism"},{"source":"ref:mickolajczyk2015","machine":"kinesin","label":"Mickolajczyk et al.","section":"sources","href":"https://doi.org/10.1073/pnas.1517638112"}]},{"id":"kinesin-back-step-cause","machine":"kinesin","kind":"qa","prompt":"Under a heavy opposing load, why does kinesin-1's free head land behind its partner instead of ahead?","answer":"The pull on the stalk cancels the small forward bias from neck-linker docking.","explanation":"The landing site is set by where the head is when it touches down. The chemistry is the same as a forward step, one ATP per step.","section":"mechanism","topic":"cycle","sources":["step:The load biases the search backward","step:It lands behind and releases ADP","mechanism:Load slows the motor","ref:carter2005"],"tags":["load","back-steps"],"difficulty":3,"url":"/machines/kinesin#mechanism","cites":[{"source":"step:The load biases the search backward","machine":"kinesin","label":"Step: The load biases the search backward","section":"mechanism"},{"source":"step:It lands behind and releases ADP","machine":"kinesin","label":"Step: It lands behind and releases ADP","section":"mechanism"},{"source":"mechanism:Load slows the motor","machine":"kinesin","label":"Step: Load slows the motor","section":"mechanism"},{"source":"ref:carter2005","machine":"kinesin","label":"Carter et al.","section":"sources","anchor":"ref-carter2005","href":"https://doi.org/10.1038/nature03528"}]},{"id":"kinesin-drift-time","machine":"kinesin","kind":"cloze","prompt":"If proteins made near the spine just drifted down a 1 m nerve cell, they would need about {{300 years}} to reach the foot.","answer":"300 years","explanation":"Diffusion time grows with the square of distance: about two weeks for 1 cm becomes about 300 years for 1 m. Kinesin motors make the trip in a few days.","section":"story","topic":"purpose","sources":["fact:K2","stop:race","ref:cbtn-diffusion"],"tags":["axon","diffusion"],"difficulty":2,"url":"/machines/kinesin#story","cites":[{"source":"fact:K2","machine":"kinesin","label":"Time for a protein to drift 1 m by diffusion: 300 years","section":"story"},{"source":"stop:race","machine":"kinesin","label":"Big picture: Drift or motor","section":"story"},{"source":"ref:cbtn-diffusion","machine":"kinesin","label":"Cell Biology by the Numbers","section":"sources","href":"https://book.bionumbers.org/what-are-the-time-scales-for-diffusion-in-cells/"}]},{"id":"kinesin-fast-transport-time","machine":"kinesin","kind":"qa","prompt":"About how long does fast outward transport take to carry cargo 1 m along a nerve cell?","answer":"About 2.5-5 days.","explanation":"Fast outward transport runs at 200-400 mm per day (2-5 µm/s).","section":"story","topic":"numbers","sources":["fact:K3","ref:brown2003"],"tags":["axon","transport"],"difficulty":2,"url":"/machines/kinesin#story","cites":[{"source":"fact:K3","machine":"kinesin","label":"Speed of fast outward transport in axons: 200–400 mm per day","section":"story"},{"source":"ref:brown2003","machine":"kinesin","label":"Brown 2003","section":"sources","href":"https://doi.org/10.1083/jcb.200212017"}]},{"id":"kinesin-teams-of-motors","machine":"kinesin","kind":"qa","prompt":"A single kinesin falls off after about 1 µm. How does cargo still finish a 1 m trip down an axon?","answer":"Teams of motors grab on again and again.","explanation":"One motor would let go about 10^6 times on the way; this is where the delivery-truck analogy breaks.","section":"story","topic":"purpose","sources":["analogy:A delivery truck on a highway","fact:K8"],"tags":["analogy","processivity"],"difficulty":2,"url":"/machines/kinesin#story","cites":[{"source":"analogy:A delivery truck on a highway","machine":"kinesin","label":"Analogy: A delivery truck on a highway","section":"story"},{"source":"fact:K8","machine":"kinesin","label":"Steps for one kinesin to cross 1 m: 1.25 × 10^8 steps, one ATP each","section":"story"}]},{"id":"kinesin-walker-analogy","machine":"kinesin","kind":"qa","prompt":"The 'walker with two feet' picture of kinesin gets hand-over-hand stepping right. What does it get wrong about the free foot?","answer":"It does not swing by muscle: it jiggles by heat, and the zipped neck linker only biases where it lands.","explanation":"Diffusion does the travel; docking sets the direction.","section":"story","topic":"purpose","sources":["analogy:A walker with two feet","step:The free head searches"],"tags":["analogy","diffusion"],"difficulty":2,"url":"/machines/kinesin#story","cites":[{"source":"analogy:A walker with two feet","machine":"kinesin","label":"Analogy: A walker with two feet","section":"story"},{"source":"step:The free head searches","machine":"kinesin","label":"Step: The free head searches","section":"mechanism"}]},{"id":"kinesin-dynein-tug","machine":"kinesin","kind":"qa","prompt":"On a cargo vesicle in an axon, which motor pulls against kinesin, back toward the cell body?","answer":"Dynein.","explanation":"Vesicles from mouse brain carry 1-4 kinesins and 1-5 dyneins, and the cargo moves in fits and starts.","section":"story","topic":"purpose","sources":["fact:K6","link:dynein","stop:vesicle","ref:hendricks2010"],"tags":["dynein","axon","cargo"],"difficulty":1,"url":"/machines/kinesin#story","cites":[{"source":"fact:K6","machine":"kinesin","label":"Kinesins on one brain vesicle: 1–4 kinesins","section":"story"},{"source":"link:dynein","machine":"kinesin","label":"Link to Dynein","section":"story"},{"source":"stop:vesicle","machine":"kinesin","label":"Big picture: Vesicle team","section":"story"},{"source":"ref:hendricks2010","machine":"kinesin","label":"Hendricks 2010","section":"sources","href":"https://doi.org/10.1016/j.cub.2010.02.058"}]},{"id":"kinesin-svoboda-method","machine":"kinesin","kind":"qa","prompt":"How did Svoboda and colleagues (1993) first see kinesin's 8 nm steps?","answer":"With optical trapping interferometry: they tracked a bead carried by one kinesin.","explanation":"The bead moved in discrete 8 nm steps, the length of one tubulin dimer, the repeat of the track.","section":"evidence","topic":"numbers","sources":["evidence:svoboda1993-8nm-steps","ref:svoboda1993"],"tags":["method","optical-trap","step"],"difficulty":2,"url":"/machines/kinesin#evidence","cites":[{"source":"evidence:svoboda1993-8nm-steps","machine":"kinesin","label":"Step size of single kinesin molecules (Svoboda K 1993)","section":"evidence","anchor":"ev-svoboda1993-8nm-steps"},{"source":"ref:svoboda1993","machine":"kinesin","label":"Svoboda et al.","section":"sources","anchor":"ref-svoboda1993","href":"https://doi.org/10.1038/365721a0"}]},{"id":"kinesin-hand-over-hand-proof","machine":"kinesin","kind":"qa","prompt":"Yildiz and colleagues put one dye on one head of kinesin. What pattern of dye movement showed that kinesin walks hand over hand?","answer":"The dye jumped about 17 nm, then not at all, in turn.","explanation":"Meanwhile the motor as a whole moved 8.3 nm per step, so the heads must swap places.","section":"evidence","topic":"numbers","sources":["evidence:yildiz2004-head-step","ref:yildiz2004"],"tags":["method","fluorescence","hand-over-hand"],"difficulty":2,"url":"/machines/kinesin#evidence","cites":[{"source":"evidence:yildiz2004-head-step","machine":"kinesin","label":"Step of a single labelled kinesin head (Yildiz A 2004)","section":"evidence","anchor":"ev-yildiz2004-head-step"},{"source":"ref:yildiz2004","machine":"kinesin","label":"Yildiz et al.","section":"sources","anchor":"ref-yildiz2004","href":"https://doi.org/10.1126/science.1093753"}]},{"id":"kinesin-one-atp-method","machine":"kinesin","kind":"qa","prompt":"How did Coy and colleagues (1999) show that kinesin takes one step per ATP?","answer":"They divided the bead speed by an 8.1 nm step, then by the ATP turnover of the same beads.","explanation":"Every preparation gave close to one: 1.08 ± 0.09 steps per ATP pooled.","section":"evidence","topic":"numbers","sources":["evidence:coy1999-steps-per-atp","ref:coy1999"],"tags":["method","atp","coupling"],"difficulty":3,"url":"/machines/kinesin#evidence","cites":[{"source":"evidence:coy1999-steps-per-atp","machine":"kinesin","label":"Steps per ATP hydrolysed, from speed and ATPase of the same bead-bound motors (Coy DL 1999)","section":"evidence","anchor":"ev-coy1999-steps-per-atp"},{"source":"ref:coy1999","machine":"kinesin","label":"Coy, Wagenbach and Howard, J Biol Chem 1999","section":"sources","href":"https://doi.org/10.1074/jbc.274.6.3667"}]},{"id":"kinesin-backstep-stall","machine":"kinesin","kind":"cloze","prompt":"In Carter and Cross's optical trap, kinesin-1 took as many back steps as forward steps near {{7 pN}} of opposing load.","answer":"7 pN","explanation":"That is the stall force; above it kinesin walked backward. It is higher than the 5.4 pN measured against a glass fibre.","section":"evidence","topic":"debate","sources":["evidence:carter2005-backstep-ratio","evidence:meyhofer1995-stall-force","ref:carter2005"],"tags":["stall-force","back-steps","load"],"difficulty":2,"url":"/machines/kinesin#evidence","cites":[{"source":"evidence:carter2005-backstep-ratio","machine":"kinesin","label":"Ratio of forward to backward steps against load (Carter NJ 2005)","section":"evidence","anchor":"ev-carter2005-backstep-ratio"},{"source":"evidence:meyhofer1995-stall-force","machine":"kinesin","label":"Maximum force of a single kinesin molecule against an elastic glass fibre (Meyhöfer E 1995)","section":"evidence","anchor":"ev-meyhofer1995-stall-force"},{"source":"ref:carter2005","machine":"kinesin","label":"Carter et al.","section":"sources","anchor":"ref-carter2005","href":"https://doi.org/10.1038/nature03528"}]},{"id":"kinesin-stall-vs-atp","machine":"kinesin","kind":"qa","prompt":"Visscher and colleagues (1999) measured kinesin's stall force at different ATP levels. What did they find?","answer":"The stall force rises with ATP concentration.","explanation":"So the stall force is not one fixed number. Against a glass fibre, Meyhöfer and Howard found about the same force at high and low ATP.","section":"evidence","topic":"debate","sources":["evidence:visscher1999-stall-vs-atp","evidence:meyhofer1995-stall-force","ref:visscher1999"],"tags":["stall-force","atp"],"difficulty":3,"url":"/machines/kinesin#evidence","cites":[{"source":"evidence:visscher1999-stall-vs-atp","machine":"kinesin","label":"Stall force as a function of ATP concentration (Visscher K 1999)","section":"evidence","anchor":"ev-visscher1999-stall-vs-atp"},{"source":"evidence:meyhofer1995-stall-force","machine":"kinesin","label":"Maximum force of a single kinesin molecule against an elastic glass fibre (Meyhöfer E 1995)","section":"evidence","anchor":"ev-meyhofer1995-stall-force"},{"source":"ref:visscher1999","machine":"kinesin","label":"Visscher et al.","section":"sources","anchor":"ref-visscher1999","href":"https://doi.org/10.1038/22146"}]},{"id":"kinesin-neck-charge","machine":"kinesin","kind":"qa","prompt":"Why does extra positive charge on kinesin's neck coiled coil make its runs longer?","answer":"The positive neck holds on to the negatively charged tails of tubulin.","explanation":"Runs grew about four-fold with speed unchanged. Salt, or removing the tubulin tails, cancelled the gain.","section":"evidence","topic":"numbers","sources":["evidence:neck-charge-thorn2000","ref:thorn2000"],"tags":["processivity","engineering"],"difficulty":3,"url":"/machines/kinesin#evidence","cites":[{"source":"evidence:neck-charge-thorn2000","machine":"kinesin","label":"Run length after changing the charge of the neck coiled coil (Thorn KS 2000)","section":"evidence","anchor":"ev-neck-charge-thorn2000"},{"source":"ref:thorn2000","machine":"kinesin","label":"Thorn et al.","section":"sources","anchor":"ref-thorn2000","href":"https://doi.org/10.1083/jcb.151.5.1093"}]},{"id":"kinesin-phosphate-evidence","machine":"kinesin","kind":"qa","prompt":"In Milic and colleagues' force-clamp runs, what effect of added phosphate showed that kinesin's processivity is gated by phosphate release?","answer":"Added phosphate made the runs longer.","explanation":"Under forward load, runs became up to about twice as long: the rear head lets go only after it releases phosphate.","section":"evidence","topic":"numbers","sources":["evidence:milic2014-run-length","step:The rear head lets go","ref:milic2014"],"tags":["processivity","phosphate","method"],"difficulty":3,"url":"/machines/kinesin#evidence","cites":[{"source":"evidence:milic2014-run-length","machine":"kinesin","label":"Unloaded run length of single kinesin molecules (Milic B 2014)","section":"evidence","anchor":"ev-milic2014-run-length"},{"source":"step:The rear head lets go","machine":"kinesin","label":"Step: The rear head lets go","section":"mechanism"},{"source":"ref:milic2014","machine":"kinesin","label":"Milic et al.","section":"sources","anchor":"ref-milic2014","href":"https://doi.org/10.1073/pnas.1410943111"}]},{"id":"myosin-class-differences","machine":"myosin","kind":"qa","prompt":"Myosin classes share the same head. Name the three things that differ between them.","answer":"Lever length, direction, and time spent bound to actin.","explanation":"Across isoforms the motor-domain states are nearly invariant; the lever position differs.","section":"summary","topic":"purpose","sources":["machine:summary","evolution:Variable lever","ref:houdusse2000"],"tags":["classes","lever"],"difficulty":2,"url":"/machines/myosin#summary","cites":[{"source":"machine:summary","machine":"myosin","label":"Summary","section":"summary"},{"source":"evolution:Variable lever","machine":"myosin","label":"Variable lever","section":"summary"},{"source":"ref:houdusse2000","machine":"myosin","label":"Houdusse et al.","section":"sources","anchor":"ref-houdusse2000","href":"https://doi.org/10.1073/pnas.200376897"}]},{"id":"myosin-converter-lever-role","machine":"myosin","kind":"qa","prompt":"In a myosin head, what does the long lever-arm helix do with the rotation of the converter?","answer":"It amplifies the rotation into a stroke.","explanation":"The converter turns with the motor state; the lever turns that small turn into a large stroke, so lever length sets the stroke.","section":"summary","topic":"parts","sources":["component:converter and lever arm"],"tags":["lever","converter"],"difficulty":1,"url":"/machines/myosin#summary","cites":[{"source":"component:converter and lever arm","machine":"myosin","label":"Part: converter and lever arm","section":"summary"}]},{"id":"myosin-tail-role","machine":"myosin","kind":"qa","prompt":"What does the coiled-coil tail of myosin do?","answer":"It joins the two heavy chains and builds thick filaments or binds cargo.","explanation":"The head does the work on actin; the tail decides what the pull is applied to.","section":"summary","topic":"parts","sources":["component:coiled-coil tail"],"tags":["tail","cargo"],"difficulty":1,"url":"/machines/myosin#summary","cites":[{"source":"component:coiled-coil tail","machine":"myosin","label":"Part: coiled-coil tail","section":"summary"}]},{"id":"myosin-duty-ratio-definition","machine":"myosin","kind":"qa","prompt":"What is the duty ratio of a myosin head?","answer":"The fraction of the cycle it spends strongly bound to actin.","explanation":"Myosin-5 has a duty ratio of about 0.7; muscle myosin spends only a small fraction of its cycle bound.","section":"summary","topic":"numbers","sources":["stat:Duty ratio, myosin-5","evolution:Duty ratio and function","ref:delacruz1999"],"tags":["duty-ratio"],"difficulty":1,"url":"/machines/myosin#summary","cites":[{"source":"stat:Duty ratio, myosin-5","machine":"myosin","label":"Key number: Duty ratio, myosin-5","section":"summary"},{"source":"evolution:Duty ratio and function","machine":"myosin","label":"Duty ratio and function","section":"summary"},{"source":"ref:delacruz1999","machine":"myosin","label":"De La Cruz et al.","section":"sources","anchor":"ref-delacruz1999","href":"https://doi.org/10.1073/pnas.96.24.13726"}]},{"id":"myosin-muscle-low-duty","machine":"myosin","kind":"qa","prompt":"Muscle myosin spends only a small fraction of its cycle bound to actin. How does muscle still pull steadily?","answer":"Its heads work in large ensembles.","explanation":"Each thick filament carries about 300 myosins. Only a few hold on at any moment; each pulls, lets go and grabs again.","section":"summary","topic":"numbers","sources":["stat:Duty ratio, myosin-5","stop:filament","ref:delacruz1999"],"tags":["duty-ratio","muscle"],"difficulty":2,"url":"/machines/myosin#summary","cites":[{"source":"stat:Duty ratio, myosin-5","machine":"myosin","label":"Key number: Duty ratio, myosin-5","section":"summary"},{"source":"stop:filament","machine":"myosin","label":"Big picture: The thick filament","section":"story"},{"source":"ref:delacruz1999","machine":"myosin","label":"De La Cruz et al.","section":"sources","anchor":"ref-delacruz1999","href":"https://doi.org/10.1073/pnas.96.24.13726"}]},{"id":"myosin-cardiac-drugs-pocket","machine":"myosin","kind":"qa","prompt":"Omecamtiv mecarbil activates cardiac myosin and mavacamten inhibits it. What do the two drugs have in common at the structure level?","answer":"They bind the same pocket.","explanation":"Structures of this shared pocket support rational design of the next cardiac myosin drugs.","section":"summary","topic":"debate","sources":["species:Beta-cardiac myosin","frontier:Cardiac myosin drugs","ref:auguin2024"],"tags":["drugs","heart"],"difficulty":2,"url":"/machines/myosin#summary","cites":[{"source":"species:Beta-cardiac myosin","machine":"myosin","label":"Beta-cardiac myosin","section":"summary"},{"source":"frontier:Cardiac myosin drugs","machine":"myosin","label":"Open question: Cardiac myosin drugs","section":"summary"},{"source":"ref:auguin2024","machine":"myosin","label":"Auguin et al.","section":"sources","anchor":"ref-auguin2024","href":"https://doi.org/10.1038/s41467-024-47587-9"}]},{"id":"myosin-cardiac-drug-approved","machine":"myosin","kind":"cloze","prompt":"Both cardiac myosin drugs completed phase 3 trials, and regulators have approved {{mavacamten}}.","answer":"mavacamten","explanation":"Mavacamten, first reported as MYK-461, lowers the ATPase of cardiac myosin.","section":"summary","topic":"debate","sources":["frontier:Cardiac myosin drugs","ref:green2016"],"tags":["drugs","heart"],"difficulty":2,"url":"/machines/myosin#summary","cites":[{"source":"frontier:Cardiac myosin drugs","machine":"myosin","label":"Open question: Cardiac myosin drugs","section":"summary"},{"source":"ref:green2016","machine":"myosin","label":"Green et al.","section":"sources","anchor":"ref-green2016","href":"https://doi.org/10.1126/science.aad3456"}]},{"id":"myosin-cleft-gates-phosphate","machine":"myosin","kind":"qa","prompt":"When a primed myosin head first docks on actin, why can phosphate not leave yet?","answer":"The actin-binding cleft is still open.","explanation":"Contacts with the actin N-terminus rotate the upper 50-kDa subdomain and close the cleft; only then can phosphate leave and the head bind strongly.","section":"mechanism","topic":"cycle","sources":["mechanism:A primed head docks on actin","mechanism:The cleft closes and phosphate leaves","ref:klebl2025"],"tags":["phosphate","cleft"],"difficulty":2,"url":"/machines/myosin#mechanism","cites":[{"source":"mechanism:A primed head docks on actin","machine":"myosin","label":"Step: A primed head docks on actin","section":"mechanism"},{"source":"mechanism:The cleft closes and phosphate leaves","machine":"myosin","label":"Step: The cleft closes and phosphate leaves","section":"mechanism"},{"source":"ref:klebl2025","machine":"myosin","label":"Klebl et al.","section":"sources","anchor":"ref-klebl2025","href":"https://doi.org/10.1038/s41586-025-08876-5"}]},{"id":"myosin-power-stroke-angle","machine":"myosin","kind":"cloze","prompt":"In the myosin power stroke, the converter rotates and the lever swings through about {{93 degrees}}, mostly along the filament.","answer":"93 degrees","explanation":"Measured for myosin-5 on actin by time-resolved cryo-EM. Because the swing runs along the filament, nearly all of it moves the load forward.","section":"mechanism","topic":"cycle","sources":["mechanism:The power stroke","stat:Lever swing on actin","ref:klebl2025"],"tags":["power-stroke","lever"],"difficulty":2,"url":"/machines/myosin#mechanism","cites":[{"source":"mechanism:The power stroke","machine":"myosin","label":"Step: The power stroke","section":"mechanism"},{"source":"stat:Lever swing on actin","machine":"myosin","label":"Key number: Lever swing on actin","section":"summary"},{"source":"ref:klebl2025","machine":"myosin","label":"Klebl et al.","section":"sources","anchor":"ref-klebl2025","href":"https://doi.org/10.1038/s41586-025-08876-5"}]},{"id":"myosin-atp-releases-why","machine":"myosin","kind":"qa","prompt":"Why does ATP binding make a myosin head let go of actin?","answer":"A head cannot hold both ATP and actin tightly, so the actin-binding cleft opens.","explanation":"Pospich and colleagues call ATP binding and actin binding reciprocal. The transducer links the pocket to the cleft.","section":"mechanism","topic":"cycle","sources":["step:The rear head lets go","mechanism:ATP frees the head","ref:pospich2021"],"tags":["atp","cleft"],"difficulty":3,"url":"/machines/myosin#mechanism","cites":[{"source":"step:The rear head lets go","machine":"myosin","label":"Step: The rear head lets go","section":"mechanism"},{"source":"mechanism:ATP frees the head","machine":"myosin","label":"Step: ATP frees the head","section":"mechanism"},{"source":"ref:pospich2021","machine":"myosin","label":"Pospich et al.","section":"sources","anchor":"ref-pospich2021","href":"https://doi.org/10.7554/eLife.73724"}]},{"id":"myosin-recovery-stroke","machine":"myosin","kind":"qa","prompt":"What does a myosin head do during the recovery stroke, off actin?","answer":"It swings its lever back and hydrolyses ATP, holding ADP and phosphate in the primed state.","explanation":"This re-cocks the lever, so the head is ready for its next power stroke.","section":"mechanism","topic":"cycle","sources":["mechanism:Recovery stroke off actin","ref:houdusse1999"],"tags":["recovery-stroke","atp"],"difficulty":2,"url":"/machines/myosin#mechanism","cites":[{"source":"mechanism:Recovery stroke off actin","machine":"myosin","label":"Step: Recovery stroke off actin","section":"mechanism"},{"source":"ref:houdusse1999","machine":"myosin","label":"Houdusse et al.","section":"sources","anchor":"ref-houdusse1999","href":"https://doi.org/10.1016/S0092-8674(00)80756-4"}]},{"id":"myosin-myosin5-pace-step","machine":"myosin","kind":"qa","prompt":"Which step sets the walking pace of myosin-5?","answer":"ADP release from the rear head.","explanation":"ADP leaves at 11.7 per second, close to the whole ATPase rate of 12–15 per second.","section":"mechanism","topic":"cycle","sources":["step:ADP leaves the rear head","evidence:delacruz1999-adp-release","ref:delacruz1999"],"tags":["adp","myosin-5","kinetics"],"difficulty":2,"url":"/machines/myosin#mechanism","cites":[{"source":"step:ADP leaves the rear head","machine":"myosin","label":"Step: ADP leaves the rear head","section":"mechanism"},{"source":"evidence:delacruz1999-adp-release","machine":"myosin","label":"ADP release from actomyosin V compared with the ATPase rate (De La Cruz EM 1999)","section":"evidence","anchor":"ev-delacruz1999-adp-release"},{"source":"ref:delacruz1999","machine":"myosin","label":"De La Cruz et al.","section":"sources","anchor":"ref-delacruz1999","href":"https://doi.org/10.1073/pnas.96.24.13726"}]},{"id":"myosin-myosin5-why-bound","machine":"myosin","kind":"qa","prompt":"Why does a single myosin-5 head spend about 70% of its cycle strongly bound to actin?","answer":"Its slowest step, ADP release, happens while it is strongly bound.","explanation":"A high duty ratio lets a two-headed myosin-5 keep one head on actin while the other steps.","section":"mechanism","topic":"cycle","sources":["step:ADP leaves the rear head","evidence:delacruz1999-duty-ratio","ref:delacruz1999"],"tags":["duty-ratio","myosin-5"],"difficulty":3,"url":"/machines/myosin#mechanism","cites":[{"source":"step:ADP leaves the rear head","machine":"myosin","label":"Step: ADP leaves the rear head","section":"mechanism"},{"source":"evidence:delacruz1999-duty-ratio","machine":"myosin","label":"Fraction of single-headed myosin V strongly bound to actin during ATP turnover (De La Cruz EM 1999)","section":"evidence","anchor":"ev-delacruz1999-duty-ratio"},{"source":"ref:delacruz1999","machine":"myosin","label":"De La Cruz et al.","section":"sources","anchor":"ref-delacruz1999","href":"https://doi.org/10.1073/pnas.96.24.13726"}]},{"id":"myosin-myosin5-13-subunits","machine":"myosin","kind":"qa","prompt":"Why do the two heads of myosin-5 bind actin 13 subunits apart?","answer":"A site 13 subunits on faces the same way, so the motor walks straight.","explanation":"Actin repeats its pattern about every 36 nm; 13 subunits at about 2.75 nm each is about 36 nm, the step of myosin-5.","section":"mechanism","topic":"cycle","sources":["step:Both heads hold actin","evidence:walker2000-head-span","ref:walker2000"],"tags":["actin","myosin-5","step"],"difficulty":3,"url":"/machines/myosin#mechanism","cites":[{"source":"step:Both heads hold actin","machine":"myosin","label":"Step: Both heads hold actin","section":"mechanism"},{"source":"evidence:walker2000-head-span","machine":"myosin","label":"Axial distance between the two heads of myosin V bound to actin (Walker ML 2000)","section":"evidence","anchor":"ev-walker2000-head-span"},{"source":"ref:walker2000","machine":"myosin","label":"Walker et al.","section":"sources","href":"https://doi.org/10.1038/35015592"}]},{"id":"myosin-myosin5-lead-held","machine":"myosin","kind":"qa","prompt":"In a two-headed myosin-5 on actin, what holds the lead head's lever in the primed position after it releases phosphate?","answer":"The pull from the rear head.","explanation":"The lead head waits with ADP until the rear head lets go; then nothing holds its lever back and it swings.","section":"mechanism","topic":"cycle","sources":["step:The new lead head releases phosphate","step:The lead head swings its lever","ref:klebl2025"],"tags":["myosin-5","strain"],"difficulty":3,"url":"/machines/myosin#mechanism","cites":[{"source":"step:The new lead head releases phosphate","machine":"myosin","label":"Step: The new lead head releases phosphate","section":"mechanism"},{"source":"step:The lead head swings its lever","machine":"myosin","label":"Step: The lead head swings its lever","section":"mechanism"},{"source":"ref:klebl2025","machine":"myosin","label":"Klebl et al.","section":"sources","anchor":"ref-klebl2025","href":"https://doi.org/10.1038/s41586-025-08876-5"}]},{"id":"myosin-muscle-layout","machine":"myosin","kind":"qa","prompt":"In muscle, how do myosins add their forces, and how do sarcomeres add their shortening?","answer":"Myosins sit side by side, so forces add; sarcomeres sit end to end, so shortenings add.","explanation":"This layout turns strokes of a few nanometres into movements of centimetres.","section":"story","topic":"purpose","sources":["story:summary"],"tags":["muscle","sarcomere"],"difficulty":2,"url":"/machines/myosin#story","cites":[{"source":"story:summary","machine":"myosin","label":"Big picture","section":"story"}]},{"id":"myosin-train-analogy-breaks","machine":"myosin","kind":"qa","prompt":"Muscle is like train carriages coupled in a row. What does this analogy get wrong about the sarcomere?","answer":"Nothing in a sarcomere shrinks; the filaments slide past each other.","explanation":"The analogy is right that units in series add their travel and units side by side add their force.","section":"story","topic":"purpose","sources":["analogy:Train carriages coupled in a row","stop:sarcomere"],"tags":["analogy","sarcomere"],"difficulty":2,"url":"/machines/myosin#story","cites":[{"source":"analogy:Train carriages coupled in a row","machine":"myosin","label":"Analogy: Train carriages coupled in a row","section":"story"},{"source":"stop:sarcomere","machine":"myosin","label":"Big picture: The sarcomere","section":"story"}]},{"id":"myosin-rowing-analogy-breaks","machine":"myosin","kind":"qa","prompt":"Myosin heads are like a rowing crew. Where does this analogy break down?","answer":"Rowers pull in time; each myosin head works on its own clock.","explanation":"Each head also lets go of actin for most of its cycle, so only a few hold on at any moment.","section":"story","topic":"purpose","sources":["analogy:A rowing crew"],"tags":["analogy","duty-ratio"],"difficulty":2,"url":"/machines/myosin#story","cites":[{"source":"analogy:A rowing crew","machine":"myosin","label":"Analogy: A rowing crew","section":"story"}]},{"id":"myosin-gain-to-hand","machine":"myosin","kind":"cloze","prompt":"From the phosphate in a myosin head to the hand, movement grows about {{30 million}} times.","answer":"30 million","explanation":"About 0.5 nm at the active site becomes about 15 mm at the hand, through the lever, many heads, sarcomeres in a row and the arm (human biceps estimate).","section":"story","topic":"numbers","sources":["fact:M9","ref:bnid107902"],"tags":["scale","gain"],"difficulty":2,"url":"/machines/myosin#story","cites":[{"source":"fact:M9","machine":"myosin","label":"Gain from the phosphate to the hand: 3 × 10^7 times","section":"story"},{"source":"ref:bnid107902","machine":"myosin","label":"BNID 107902","section":"sources","href":"https://bionumbers.hms.harvard.edu/bionumber.aspx?id=107902"}]},{"id":"myosin-link-serca","machine":"myosin","kind":"qa","prompt":"Calcium switches myosin on. How does SERCA switch it off?","answer":"By pumping the calcium away.","explanation":"Calcium is the on switch for myosin, so removing it switches myosin off.","section":"story","topic":"purpose","sources":["link:serca"],"tags":["serca","calcium"],"difficulty":1,"url":"/machines/myosin#story","cites":[{"source":"link:serca","machine":"myosin","label":"Link to SERCA calcium pump","section":"story"}]},{"id":"myosin-kinesin-shared-core","machine":"myosin","kind":"qa","prompt":"Myosin and kinesin share almost no sequence identity. What do they share?","answer":"The same fold of the catalytic core.","explanation":"The two motor families probably evolved from a common ancestor; kinesin walks on microtubules, myosin on actin.","section":"story","topic":"purpose","sources":["evolution:Common core with kinesin","link:kinesin","ref:kull1996"],"tags":["kinesin","evolution"],"difficulty":2,"url":"/machines/myosin#story","cites":[{"source":"evolution:Common core with kinesin","machine":"myosin","label":"Common core with kinesin","section":"summary"},{"source":"link:kinesin","machine":"myosin","label":"Link to Kinesin","section":"story"},{"source":"ref:kull1996","machine":"myosin","label":"Kull et al.","section":"sources","anchor":"ref-kull1996","href":"https://doi.org/10.1038/380550a0"}]},{"id":"myosin-finer-method","machine":"myosin","kind":"qa","prompt":"How did Finer, Simmons and Spudich (1994) measure the step of a single myosin molecule?","answer":"With a laser trap holding one actin filament against a single myosin molecule.","explanation":"Each time the myosin bound, the filament jumped forward in a discrete step, averaging 11 nm at low load.","section":"evidence","topic":"numbers","sources":["evidence:finer1994-step","ref:finer1994"],"tags":["optical-trap","stroke"],"difficulty":2,"url":"/machines/myosin#evidence","cites":[{"source":"evidence:finer1994-step","machine":"myosin","label":"Displacement produced by a single myosin molecule at low load (Finer JT 1994)","section":"evidence","anchor":"ev-finer1994-step"},{"source":"ref:finer1994","machine":"myosin","label":"Finer et al.","section":"sources","anchor":"ref-finer1994","href":"https://doi.org/10.1038/368113a0"}]},{"id":"myosin-stroke-dispute","machine":"myosin","kind":"qa","prompt":"Finer 1994 found a stroke of about 11 nm for muscle myosin. What stroke did Molloy 1995 find for a single head (S1)?","answer":"About 4 nm.","explanation":"The single-head estimate depends on how the broad spread of displacements is analysed, so the stroke size is disputed.","section":"evidence","topic":"debate","sources":["evidence:molloy1995-single-head-stroke","stat:Stroke per head, muscle myosin"],"tags":["stroke","disputed"],"difficulty":2,"url":"/machines/myosin#evidence","cites":[{"source":"evidence:molloy1995-single-head-stroke","machine":"myosin","label":"Working stroke and force of a single myosin head (S1) (Molloy JE 1995)","section":"evidence","anchor":"ev-molloy1995-single-head-stroke"},{"source":"stat:Stroke per head, muscle myosin","machine":"myosin","label":"Key number: Stroke per head, muscle myosin","section":"summary"}]},{"id":"myosin-yildiz-hand-over-hand","machine":"myosin","kind":"qa","prompt":"Yildiz and colleagues tracked one dye on the lever of myosin-5. What step pattern showed that it walks hand over hand?","answer":"Long and short steps in turn (37 + 2x and 37 − 2x nm).","explanation":"If the same head always led, as in an inchworm, every step would be the same size. The centre of mass moves about 37 nm per ATP.","section":"evidence","topic":"numbers","sources":["evidence:yildiz2003-hand-over-hand","ref:yildiz2003"],"tags":["myosin-5","stepping"],"difficulty":3,"url":"/machines/myosin#evidence","cites":[{"source":"evidence:yildiz2003-hand-over-hand","machine":"myosin","label":"Step size of a single dye on the myosin V light-chain domain (Yildiz A 2003)","section":"evidence","anchor":"ev-yildiz2003-hand-over-hand"},{"source":"ref:yildiz2003","machine":"myosin","label":"Yildiz et al.","section":"sources","anchor":"ref-yildiz2003","href":"https://doi.org/10.1126/science.1084398"}]},{"id":"myosin-uyeda-lever-test","machine":"myosin","kind":"qa","prompt":"What did Uyeda, Abramson and Spudich (1996) find when they changed the neck length of Dictyostelium myosin?","answer":"Gliding speed rose linearly with lever length.","explanation":"Speed fell on a line that reached zero near a fulcrum in the motor domain, so the neck acts as a lever arm.","section":"evidence","topic":"numbers","sources":["evidence:uyeda1996-velocity-vs-lever","stat:Velocity versus lever length","ref:uyeda1996"],"tags":["lever","motility"],"difficulty":2,"url":"/machines/myosin#evidence","cites":[{"source":"evidence:uyeda1996-velocity-vs-lever","machine":"myosin","label":"Actin sliding velocity against lever-arm length (Uyeda TQ 1996)","section":"evidence","anchor":"ev-uyeda1996-velocity-vs-lever"},{"source":"stat:Velocity versus lever length","machine":"myosin","label":"Key number: Velocity versus lever length","section":"summary"},{"source":"ref:uyeda1996","machine":"myosin","label":"Uyeda et al.","section":"sources","anchor":"ref-uyeda1996","href":"https://doi.org/10.1073/pnas.93.9.4459"}]},{"id":"myosin-delacruz-method","machine":"myosin","kind":"qa","prompt":"How did De La Cruz and colleagues measure how much of the cycle a myosin-5 head stays strongly bound?","answer":"With pyrene-labelled actin, whose glow is quenched by strongly bound myosin, in stopped flow.","explanation":"Most heads stayed strongly bound for most of each cycle: a duty ratio of about 0.7.","section":"evidence","topic":"numbers","sources":["evidence:delacruz1999-duty-ratio","ref:delacruz1999"],"tags":["duty-ratio","stopped-flow"],"difficulty":3,"url":"/machines/myosin#evidence","cites":[{"source":"evidence:delacruz1999-duty-ratio","machine":"myosin","label":"Fraction of single-headed myosin V strongly bound to actin during ATP turnover (De La Cruz EM 1999)","section":"evidence","anchor":"ev-delacruz1999-duty-ratio"},{"source":"ref:delacruz1999","machine":"myosin","label":"De La Cruz et al.","section":"sources","anchor":"ref-delacruz1999","href":"https://doi.org/10.1073/pnas.96.24.13726"}]},{"id":"myosin-rief-adp-gates","machine":"myosin","kind":"qa","prompt":"In single-molecule stepping of myosin-5 (Rief 2000), what showed that ADP release gates each step?","answer":"Adding ADP slowed the stepping rate, from 12.5 to 6.4 per second.","explanation":"The rate matches ADP release measured in solution by De La Cruz and colleagues.","section":"evidence","topic":"numbers","sources":["evidence:rief2000-dwell-rate","ref:rief2000"],"tags":["adp","optical-trap"],"difficulty":3,"url":"/machines/myosin#evidence","cites":[{"source":"evidence:rief2000-dwell-rate","machine":"myosin","label":"Rate-limiting transition between myosin-V steps (Rief M 2000)","section":"evidence","anchor":"ev-rief2000-dwell-rate"},{"source":"ref:rief2000","machine":"myosin","label":"Rief et al.","section":"sources","anchor":"ref-rief2000","href":"https://doi.org/10.1073/pnas.97.17.9482"}]},{"id":"myosin-klebl-method","machine":"myosin","kind":"qa","prompt":"How did Klebl and colleagues (2025) see myosin-5 both before and after its stroke on actin?","answer":"They mixed myosin-ADP-Pi with actin and froze it 10 or 120 ms later (time-resolved cryo-EM).","explanation":"Primed motors fell from 62% at 10 ms to 36% at 120 ms, so both shapes could be compared on the same filament.","section":"evidence","topic":"numbers","sources":["evidence:lever-swing-klebl2025","ref:klebl2025"],"tags":["cryo-em","power-stroke"],"difficulty":2,"url":"/machines/myosin#evidence","cites":[{"source":"evidence:lever-swing-klebl2025","machine":"myosin","label":"Lever swing of myosin-5 on actin, primed to post-power stroke (Klebl DP 2025)","section":"evidence","anchor":"ev-lever-swing-klebl2025"},{"source":"ref:klebl2025","machine":"myosin","label":"Klebl et al.","section":"sources","anchor":"ref-klebl2025","href":"https://doi.org/10.1038/s41586-025-08876-5"}]},{"id":"dynein-direction","machine":"dynein","kind":"cloze","prompt":"Cytoplasmic dynein carries cargo along microtubules toward the {{minus end}}.","answer":"minus end","explanation":"Kinesin walks the same microtubule tracks the other way, so the two motors pull cargo in a tug of war.","section":"summary","topic":"purpose","sources":["machine:summary","machine:tagline","link:kinesin"],"tags":["direction","transport"],"difficulty":1,"url":"/machines/dynein#summary","cites":[{"source":"machine:summary","machine":"dynein","label":"Summary","section":"summary"},{"source":"machine:tagline","machine":"dynein","label":"Summary","section":"summary"},{"source":"link:kinesin","machine":"dynein","label":"Link to Kinesin","section":"story"}]},{"id":"dynein-activators","machine":"dynein","kind":"qa","prompt":"Alone, human dynein-1 is mostly inactive. Which two partners switch it on?","answer":"Dynactin and a cargo adaptor (such as BICD2).","explanation":"Together they turn dynein into an ultraprocessive motor. The adaptor also links dynein and dynactin to the cargo.","section":"summary","topic":"parts","sources":["machine:summary","component:dynactin","component:cargo adaptor","species:Human dynein-1","ref:mckenney2014"],"tags":["regulation","dynactin"],"difficulty":1,"url":"/machines/dynein#summary","cites":[{"source":"machine:summary","machine":"dynein","label":"Summary","section":"summary"},{"source":"component:dynactin","machine":"dynein","label":"Part: dynactin","section":"summary"},{"source":"component:cargo adaptor","machine":"dynein","label":"Part: cargo adaptor","section":"summary"},{"source":"species:Human dynein-1","machine":"dynein","label":"Human dynein-1","section":"summary"},{"source":"ref:mckenney2014","machine":"dynein","label":"McKenney et al.","section":"sources","anchor":"ref-mckenney2014","href":"https://doi.org/10.1126/science.1254198"}]},{"id":"dynein-ring","machine":"dynein","kind":"qa","prompt":"What forms the ring at the core of each dynein motor domain?","answer":"Six AAA+ domains (AAA1 to AAA6).","explanation":"The AAA+ fold is an ATP-binding module found in many cellular machines. In dynein, all six come in tandem from one heavy chain.","section":"summary","topic":"parts","sources":["stat:AAA+ domains in the ring","evolution:AAA+ family member","evidence:carter2011-six-aaa-domains","ref:uniprotQ14204"],"tags":["aaa","structure"],"difficulty":1,"url":"/machines/dynein#summary","cites":[{"source":"stat:AAA+ domains in the ring","machine":"dynein","label":"Key number: AAA+ domains in the ring","section":"summary"},{"source":"evolution:AAA+ family member","machine":"dynein","label":"AAA+ family member","section":"summary"},{"source":"evidence:carter2011-six-aaa-domains","machine":"dynein","label":"Number of AAA+ domains in the motor ring (Carter AP 2011)","section":"evidence","anchor":"ev-carter2011-six-aaa-domains"},{"source":"ref:uniprotQ14204","machine":"dynein","label":"UniProt Q14204, Cytoplasmic dynein 1 heavy chain 1 (human)","section":"sources","anchor":"ref-uniprotQ14204","href":"https://rest.uniprot.org/uniprotkb/Q14204"}]},{"id":"dynein-main-atp-site","machine":"dynein","kind":"qa","prompt":"Which of dynein's six AAA+ domains is the main site that splits ATP?","answer":"AAA1","explanation":"AAA2 to AAA4 also bind nucleotide, but they tune the cycle rather than drive it.","section":"summary","topic":"parts","sources":["machine:energy","step:ATP binds the rear head"],"tags":["atp","aaa"],"difficulty":2,"url":"/machines/dynein#summary","cites":[{"source":"machine:energy","machine":"dynein","label":"Summary","section":"summary"},{"source":"step:ATP binds the rear head","machine":"dynein","label":"Step: ATP binds the rear head","section":"mechanism"}]},{"id":"dynein-stalk","machine":"dynein","kind":"cloze","prompt":"Dynein's ATPase ring reaches the microtubule through a coiled-coil {{stalk}} about 15 nm long, with the microtubule-binding foot (MTBD) at its tip.","answer":"stalk","explanation":"Ring and foot are 15 nm apart, so signals between them must travel along this rod.","section":"summary","topic":"parts","sources":["stat:Stalk length","component:MTBD","machine:summary","ref:carter2008"],"tags":["stalk","structure"],"difficulty":1,"url":"/machines/dynein#summary","cites":[{"source":"stat:Stalk length","machine":"dynein","label":"Key number: Stalk length","section":"summary"},{"source":"component:MTBD","machine":"dynein","label":"Part: MTBD","section":"summary"},{"source":"machine:summary","machine":"dynein","label":"Summary","section":"summary"},{"source":"ref:carter2008","machine":"dynein","label":"Carter et al.","section":"sources","anchor":"ref-carter2008","href":"https://doi.org/10.1126/science.1164424"}]},{"id":"dynein-step-size","machine":"dynein","kind":"cloze","prompt":"The most frequent step of yeast dynein is {{8 nm}}, close to the spacing of tubulin dimers.","answer":"8 nm","explanation":"Steps vary from 4 to 24 nm, and side and backward steps also occur.","section":"summary","topic":"numbers","sources":["stat:Most frequent step","evidence:reckpeterson2006-step-size","ref:reckpeterson2006"],"tags":["stepping"],"difficulty":1,"url":"/machines/dynein#summary","cites":[{"source":"stat:Most frequent step","machine":"dynein","label":"Key number: Most frequent step","section":"summary"},{"source":"evidence:reckpeterson2006-step-size","machine":"dynein","label":"Step size of the dynein dimer (centre of mass) (Reck-Peterson SL 2006)","section":"evidence","anchor":"ev-reckpeterson2006-step-size"},{"source":"ref:reckpeterson2006","machine":"dynein","label":"Reck-Peterson et al.","section":"sources","anchor":"ref-reckpeterson2006","href":"https://doi.org/10.1016/j.cell.2006.05.046"}]},{"id":"dynein-reverse-direction","machine":"dynein","kind":"qa","prompt":"How did Can and colleagues make yeast dynein walk toward the plus end?","answer":"They changed the angle and the length of its stalk.","explanation":"So stalk geometry sets dynein's direction. All natural dyneins studied so far walk toward the minus end.","section":"summary","topic":"debate","sources":["frontier:Plus-end-directed dynein","evolution:Conserved direction","evidence:can2019-plus-end-dynein","ref:can2019"],"tags":["direction","engineering"],"difficulty":2,"url":"/machines/dynein#summary","cites":[{"source":"frontier:Plus-end-directed dynein","machine":"dynein","label":"Open question: Plus-end-directed dynein","section":"summary"},{"source":"evolution:Conserved direction","machine":"dynein","label":"Conserved direction","section":"summary"},{"source":"evidence:can2019-plus-end-dynein","machine":"dynein","label":"Direction and step size after stalk length and angle changes (Can S 2019)","section":"evidence","anchor":"ev-can2019-plus-end-dynein"},{"source":"ref:can2019","machine":"dynein","label":"Can et al.","section":"sources","anchor":"ref-can2019","href":"https://doi.org/10.1038/s41586-019-0914-z"}]},{"id":"dynein-atp-closes-ring","machine":"dynein","kind":"cloze","prompt":"When ATP binds AAA1, dynein's ring of six AAA+ domains {{closes}}.","answer":"closes","explanation":"ATP binding pulls AAA1 and AAA2 together. Because the six domains form one ring, the closure spreads all the way around it.","section":"mechanism","topic":"cycle","sources":["mechanism:ATP closes the ring","step:ATP binds the rear head","ref:schmidt2015"],"tags":["atp","ring"],"difficulty":1,"url":"/machines/dynein#mechanism","cites":[{"source":"mechanism:ATP closes the ring","machine":"dynein","label":"Step: ATP closes the ring","section":"mechanism"},{"source":"step:ATP binds the rear head","machine":"dynein","label":"Step: ATP binds the rear head","section":"mechanism"},{"source":"ref:schmidt2015","machine":"dynein","label":"Schmidt et al.","section":"sources","anchor":"ref-schmidt2015","href":"https://doi.org/10.1038/nature14023"}]},{"id":"dynein-helix-sliding","machine":"dynein","kind":"cloze","prompt":"Dynein's stalk carries the signal from the ring to the foot by helix {{sliding}}, not by bending.","answer":"sliding","explanation":"As the ring closes, the buttress drags helix CC2 past CC1 by about one turn of helix, and the foot lets go of the microtubule.","section":"mechanism","topic":"cycle","sources":["step:The stalk slides and the foot lets go","mechanism:ATP closes the ring","ref:kon2009"],"tags":["stalk","signalling"],"difficulty":2,"url":"/machines/dynein#mechanism","cites":[{"source":"step:The stalk slides and the foot lets go","machine":"dynein","label":"Step: The stalk slides and the foot lets go","section":"mechanism"},{"source":"mechanism:ATP closes the ring","machine":"dynein","label":"Step: ATP closes the ring","section":"mechanism"},{"source":"ref:kon2009","machine":"dynein","label":"Kon et al.","section":"sources","anchor":"ref-kon2009","href":"https://doi.org/10.1038/nsmb.1555"}]},{"id":"dynein-registry-affinity","machine":"dynein","kind":"qa","prompt":"In dynein's stalk, what does the registry (the alignment) of the two helices CC1 and CC2 control?","answer":"How tightly the foot (MTBD) binds the microtubule.","explanation":"With AAA1 empty, the ring holds the alpha registry and the foot binds tightly. Locking the registry with disulfides traps strong or weak binding.","section":"mechanism","topic":"cycle","sources":["step:The stalk slides and the foot lets go","step:Both heads hold the microtubule","ref:kon2009"],"tags":["stalk","affinity"],"difficulty":2,"url":"/machines/dynein#mechanism","cites":[{"source":"step:The stalk slides and the foot lets go","machine":"dynein","label":"Step: The stalk slides and the foot lets go","section":"mechanism"},{"source":"step:Both heads hold the microtubule","machine":"dynein","label":"Step: Both heads hold the microtubule","section":"mechanism"},{"source":"ref:kon2009","machine":"dynein","label":"Kon et al.","section":"sources","anchor":"ref-kon2009","href":"https://doi.org/10.1038/nsmb.1555"}]},{"id":"dynein-why-linker-bends","machine":"dynein","kind":"qa","prompt":"After ATP binds, why is dynein's linker forced into a bent, primed shape?","answer":"The closed ring clashes with the straight linker; there is no room for it.","explanation":"The bent linker's free end swings across the ring toward AAA2, ready for the next power stroke.","section":"mechanism","topic":"cycle","sources":["mechanism:The ring primes the linker","step:The linker bends: the priming stroke","ref:schmidt2015"],"tags":["linker","priming"],"difficulty":2,"url":"/machines/dynein#mechanism","cites":[{"source":"mechanism:The ring primes the linker","machine":"dynein","label":"Step: The ring primes the linker","section":"mechanism"},{"source":"step:The linker bends: the priming stroke","machine":"dynein","label":"Step: The linker bends: the priming stroke","section":"mechanism"},{"source":"ref:schmidt2015","machine":"dynein","label":"Schmidt et al.","section":"sources","anchor":"ref-schmidt2015","href":"https://doi.org/10.1038/nature14023"}]},{"id":"dynein-power-stroke","machine":"dynein","kind":"cloze","prompt":"When dynein's primed head rebinds the microtubule, phosphate leaves and the linker {{straightens}}: this is the power stroke.","answer":"straightens","explanation":"The energy stored in the primed linker is released while the foot holds the track, so the tail and its cargo move toward the minus end.","section":"mechanism","topic":"cycle","sources":["mechanism:Rebinding triggers the power stroke","step:Phosphate leaves and the linker straightens","ref:chai2025"],"tags":["linker","power-stroke"],"difficulty":1,"url":"/machines/dynein#mechanism","cites":[{"source":"mechanism:Rebinding triggers the power stroke","machine":"dynein","label":"Step: Rebinding triggers the power stroke","section":"mechanism"},{"source":"step:Phosphate leaves and the linker straightens","machine":"dynein","label":"Step: Phosphate leaves and the linker straightens","section":"mechanism"},{"source":"ref:chai2025","machine":"dynein","label":"Chai et al.","section":"sources","anchor":"ref-chai2025","href":"https://doi.org/10.1038/s41594-025-01543-3"}]},{"id":"dynein-slow-step","machine":"dynein","kind":"qa","prompt":"Which step of the dynein cycle appears to be the slow one?","answer":"ADP release from AAA1.","explanation":"Among active human dynein-1 motors off microtubules, most held ADP in an open AAA1 pocket. Microtubule binding opens the pocket wide and speeds ADP release.","section":"mechanism","topic":"cycle","sources":["step:ADP leaves the new lead head","evidence:chai2025-cycle-states","ref:chai2025"],"tags":["adp","kinetics"],"difficulty":3,"url":"/machines/dynein#mechanism","cites":[{"source":"step:ADP leaves the new lead head","machine":"dynein","label":"Step: ADP leaves the new lead head","section":"mechanism"},{"source":"evidence:chai2025-cycle-states","machine":"dynein","label":"Conformational states of the human dynein-1 motor during its cycle (Chai P 2025)","section":"evidence","anchor":"ev-chai2025-cycle-states"},{"source":"ref:chai2025","machine":"dynein","label":"Chai et al.","section":"sources","anchor":"ref-chai2025","href":"https://doi.org/10.1038/s41594-025-01543-3"}]},{"id":"dynein-tension-coordination","machine":"dynein","kind":"qa","prompt":"Dynein's two heads step mostly independently, but they show coordination when far apart. What kind of mechanism does that point to?","answer":"A tension-based mechanism.","explanation":"There is no tight gate between the heads: either head can step, whether it is in front or behind.","section":"mechanism","topic":"cycle","sources":["mechanism:Two heads step loosely","evidence:dewitt2012-interhead-separation","ref:dewitt2012","ref:qiu2012"],"tags":["stepping","coordination"],"difficulty":2,"url":"/machines/dynein#mechanism","cites":[{"source":"mechanism:Two heads step loosely","machine":"dynein","label":"Step: Two heads step loosely","section":"mechanism"},{"source":"evidence:dewitt2012-interhead-separation","machine":"dynein","label":"Distance between the two heads during walking (DeWitt MA 2012)","section":"evidence","anchor":"ev-dewitt2012-interhead-separation"},{"source":"ref:dewitt2012","machine":"dynein","label":"DeWitt et al.","section":"sources","anchor":"ref-dewitt2012","href":"https://doi.org/10.1126/science.1215804"},{"source":"ref:qiu2012","machine":"dynein","label":"Qiu et al.","section":"sources","anchor":"ref-qiu2012","href":"https://doi.org/10.1038/nsmb.2205"}]},{"id":"dynein-stroke-order","machine":"dynein","kind":"qa","prompt":"In the classic dynein cycle, the head rebinds the microtubule before its linker straightens. What did Chai and colleagues see that does not fit this order?","answer":"The linker straightened even in motors that were not bound to microtubules.","explanation":"They propose instead that the cargo moves as the docked linker shifts from AAA4 to AAA5.","section":"mechanism","topic":"debate","sources":["step:Phosphate leaves and the linker straightens","ref:chai2025"],"tags":["linker","power-stroke"],"difficulty":3,"url":"/machines/dynein#mechanism","cites":[{"source":"step:Phosphate leaves and the linker straightens","machine":"dynein","label":"Step: Phosphate leaves and the linker straightens","section":"mechanism"},{"source":"ref:chai2025","machine":"dynein","label":"Chai et al.","section":"sources","anchor":"ref-chai2025","href":"https://doi.org/10.1038/s41594-025-01543-3"}]},{"id":"dynein-mucus-escalator","machine":"dynein","kind":"qa","prompt":"Dynein-driven cilia line your airways. What do they push up toward your throat?","answer":"A thin layer of mucus that traps dust and germs.","explanation":"This mucus escalator moves about 5.5 mm per minute and keeps the lungs clean without you noticing.","section":"story","topic":"purpose","sources":["stop:escalator","fact:D2","story:question","ref:bustamantemarin2017"],"tags":["cilia","lungs"],"difficulty":1,"url":"/machines/dynein#story","cites":[{"source":"stop:escalator","machine":"dynein","label":"Big picture: The mucus escalator","section":"story"},{"source":"fact:D2","machine":"dynein","label":"Speed of the mucus layer: 5.5 mm per minute","section":"story"},{"source":"story:question","machine":"dynein","label":"Big picture","section":"story"},{"source":"ref:bustamantemarin2017","machine":"dynein","label":"Bustamante-Marin 2017","section":"sources","href":"https://doi.org/10.1101/cshperspect.a028241"}]},{"id":"dynein-lung-cilia-count","machine":"dynein","kind":"cloze","prompt":"An adult human lung holds an estimated {{three trillion}} motile cilia, each driven by dynein.","answer":"three trillion","explanation":"Each cilium beats 10 to 20 times a second, which adds up to 0.9 to 1.7 million beats a day.","section":"story","topic":"numbers","sources":["fact:D1","fact:D6","story:summary","ref:bustamantemarin2017"],"tags":["cilia","scale"],"difficulty":2,"url":"/machines/dynein#story","cites":[{"source":"fact:D1","machine":"dynein","label":"Motile cilia in an adult lung: 3 × 10^12","section":"story"},{"source":"fact:D6","machine":"dynein","label":"Beats of one cilium per day: 0.9–1.7 million","section":"story"},{"source":"story:summary","machine":"dynein","label":"Big picture","section":"story"},{"source":"ref:bustamantemarin2017","machine":"dynein","label":"Bustamante-Marin 2017","section":"sources","href":"https://doi.org/10.1101/cshperspect.a028241"}]},{"id":"dynein-sliding-to-bending","machine":"dynein","kind":"qa","prompt":"Dynein arms try to slide one rail of a cilium's axoneme past the next. Why does the cilium bend instead?","answer":"The rails are tied together at the base, so they cannot slide far.","explanation":"Without the base links the rails would just slide apart; tied together, the sliding turns into a bend.","section":"story","topic":"purpose","sources":["stop:sliding","stop:axoneme"],"tags":["cilia","bending"],"difficulty":2,"url":"/machines/dynein#story","cites":[{"source":"stop:sliding","machine":"dynein","label":"Big picture: Sliding becomes bending","section":"story"},{"source":"stop:axoneme","machine":"dynein","label":"Big picture: The axoneme","section":"story"}]},{"id":"dynein-pcd","machine":"dynein","kind":"qa","prompt":"In primary ciliary dyskinesia, airway cilia lack their dynein arms. What happens to the mucus?","answer":"The cilia cannot move, so the mucus does not clear.","explanation":"The disease affects about 1 in 15,000 births, and about half of the people affected have mirrored organs.","section":"story","topic":"purpose","sources":["fact:D13","fact:D14","ref:afzelius1976","ref:bustamantemarin2017"],"tags":["disease","cilia"],"difficulty":1,"url":"/machines/dynein#story","cites":[{"source":"fact:D13","machine":"dynein","label":"Cilia in primary ciliary dyskinesia: Immotile no dynein arms","section":"story"},{"source":"fact:D14","machine":"dynein","label":"Births with primary ciliary dyskinesia: 1 in 15,000 live births","section":"story"},{"source":"ref:afzelius1976","machine":"dynein","label":"Afzelius 1976","section":"sources","href":"https://doi.org/10.1126/science.1084576"},{"source":"ref:bustamantemarin2017","machine":"dynein","label":"Bustamante-Marin 2017","section":"sources","href":"https://doi.org/10.1101/cshperspect.a028241"}]},{"id":"dynein-bimetal-breaks","machine":"dynein","kind":"qa","prompt":"A cilium is like a bimetal strip: two tied layers turn a change in length into a bend. Where does that analogy break down?","answer":"In a cilium, motors actively slide one rail along the next; in a bimetal strip, heat changes the length.","explanation":"The geometry fits, but the cause differs: the dynein arms on one rail walk along the next rail.","section":"story","topic":"purpose","sources":["analogy:A bimetal strip","stop:sliding"],"tags":["analogy","cilia"],"difficulty":2,"url":"/machines/dynein#story","cites":[{"source":"analogy:A bimetal strip","machine":"dynein","label":"Analogy: A bimetal strip","section":"story"},{"source":"stop:sliding","machine":"dynein","label":"Big picture: Sliding becomes bending","section":"story"}]},{"id":"dynein-arms-per-cilium","machine":"dynein","kind":"qa","prompt":"By calculation, about how many outer dynein arms does one airway cilium hold?","answer":"About 2,000 to 2,600.","explanation":"Four outer arms sit in each 96 nm repeat along nine doublets, over 5.5 to 7 µm of length. It is a calculation, not a measured count.","section":"story","topic":"numbers","sources":["fact:D8","fact:D7","fact:D5"],"tags":["cilia","scale"],"difficulty":3,"url":"/machines/dynein#story","cites":[{"source":"fact:D8","machine":"dynein","label":"Outer dynein arms in one cilium: 2,000–2,600","section":"story"},{"source":"fact:D7","machine":"dynein","label":"Layout of the axoneme: 9 + 2 nine doublet rails around a central pair","section":"story"},{"source":"fact:D5","machine":"dynein","label":"Length of one cilium: 6.5–7 µm","section":"story"}]},{"id":"dynein-step-method","machine":"dynein","kind":"qa","prompt":"How did Reck-Peterson and colleagues measure the 8 nm step of yeast dynein?","answer":"They tracked a quantum dot on the dynein tail to a few nanometres (FIONA, TIRF microscopy) at low ATP.","explanation":"Low ATP (4 µM) slowed stepping so single steps could be found: 1342 steps from 27 molecules.","section":"evidence","topic":"numbers","sources":["evidence:reckpeterson2006-step-size","ref:reckpeterson2006"],"tags":["method","stepping"],"difficulty":2,"url":"/machines/dynein#evidence","cites":[{"source":"evidence:reckpeterson2006-step-size","machine":"dynein","label":"Step size of the dynein dimer (centre of mass) (Reck-Peterson SL 2006)","section":"evidence","anchor":"ev-reckpeterson2006-step-size"},{"source":"ref:reckpeterson2006","machine":"dynein","label":"Reck-Peterson et al.","section":"sources","anchor":"ref-reckpeterson2006","href":"https://doi.org/10.1016/j.cell.2006.05.046"}]},{"id":"dynein-head-vs-tail-step","machine":"dynein","kind":"qa","prompt":"A label on one head of yeast dynein moved about 16 to 18 nm per step, twice as far as the tail's 8 nm. Why?","answer":"The two heads take turns stepping.","explanation":"Each head moves about two tubulin dimers each time it steps, while the tail between them advances one dimer per step.","section":"evidence","topic":"numbers","sources":["evidence:reckpeterson2006-step-size","step:It binds 16 nm ahead","ref:reckpeterson2006"],"tags":["stepping","heads"],"difficulty":2,"url":"/machines/dynein#evidence","cites":[{"source":"evidence:reckpeterson2006-step-size","machine":"dynein","label":"Step size of the dynein dimer (centre of mass) (Reck-Peterson SL 2006)","section":"evidence","anchor":"ev-reckpeterson2006-step-size"},{"source":"step:It binds 16 nm ahead","machine":"dynein","label":"Step: It binds 16 nm ahead","section":"mechanism"},{"source":"ref:reckpeterson2006","machine":"dynein","label":"Reck-Peterson et al.","section":"sources","anchor":"ref-reckpeterson2006","href":"https://doi.org/10.1016/j.cell.2006.05.046"}]},{"id":"dynein-superstall","machine":"dynein","kind":"cloze","prompt":"Pulled backward with more than its stall force, yeast dynein walks {{toward the plus end}}.","answer":"toward the plus end","explanation":"In an optical trap at 10 pN it moved about 15 nm/s toward the plus end at any ATP level, and it stepped even with no ATP.","section":"evidence","topic":"numbers","sources":["evidence:gennerich2007-superstall-backward","stat:Stall force, yeast dynein","ref:gennerich2007"],"tags":["force","optical-trap"],"difficulty":2,"url":"/machines/dynein#evidence","cites":[{"source":"evidence:gennerich2007-superstall-backward","machine":"dynein","label":"Backward walking above the stall force (Gennerich A 2007)","section":"evidence","anchor":"ev-gennerich2007-superstall-backward"},{"source":"stat:Stall force, yeast dynein","machine":"dynein","label":"Key number: Stall force, yeast dynein","section":"summary"},{"source":"ref:gennerich2007","machine":"dynein","label":"Gennerich et al.","section":"sources","anchor":"ref-gennerich2007","href":"https://doi.org/10.1016/j.cell.2007.10.016"}]},{"id":"dynein-stall-disagreement","machine":"dynein","kind":"cloze","prompt":"Reported stall forces for full-length yeast dynein differ about {{twofold}}: 7 pN (Gennerich 2007) versus 3.6 pN (Belyy 2016).","answer":"twofold","explanation":"Both values are for full-length yeast dynein. Human dynein-1 alone stalled lower still, at about 2.0 pN.","section":"evidence","topic":"debate","sources":["stat:Stall force, yeast dynein","evidence:gennerich2007-stall-force","evidence:belyy2016-human-dynein-alone","ref:gennerich2007","ref:belyy2016"],"tags":["force","contested"],"difficulty":3,"url":"/machines/dynein#evidence","cites":[{"source":"stat:Stall force, yeast dynein","machine":"dynein","label":"Key number: Stall force, yeast dynein","section":"summary"},{"source":"evidence:gennerich2007-stall-force","machine":"dynein","label":"Stall force of yeast dynein (Gennerich A 2007)","section":"evidence","anchor":"ev-gennerich2007-stall-force"},{"source":"evidence:belyy2016-human-dynein-alone","machine":"dynein","label":"Stall force of human dynein without dynactin or adaptor (Belyy V 2016)","section":"evidence","anchor":"ev-belyy2016-human-dynein-alone"},{"source":"ref:gennerich2007","machine":"dynein","label":"Gennerich et al.","section":"sources","anchor":"ref-gennerich2007","href":"https://doi.org/10.1016/j.cell.2007.10.016"},{"source":"ref:belyy2016","machine":"dynein","label":"Belyy et al.","section":"sources","anchor":"ref-belyy2016","href":"https://doi.org/10.1038/ncb3393"}]},{"id":"dynein-ddb-trap-method","machine":"dynein","kind":"qa","prompt":"How did Belyy and colleagues make their optical trap pull only on complete dynein–dynactin–BICD2 complexes?","answer":"They attached the bead through a GFP tag on the adaptor, BICD2N.","explanation":"These complexes stalled at 4.3 pN, about twice the 2.0 pN of human dynein alone.","section":"evidence","topic":"numbers","sources":["evidence:belyy2016-ddb-stall-force","stat:Stall force, human dynein-dynactin-BICD2","ref:belyy2016"],"tags":["method","force","optical-trap"],"difficulty":2,"url":"/machines/dynein#evidence","cites":[{"source":"evidence:belyy2016-ddb-stall-force","machine":"dynein","label":"Stall force of the dynein-dynactin-BICD2N complex (Belyy V 2016)","section":"evidence","anchor":"ev-belyy2016-ddb-stall-force"},{"source":"stat:Stall force, human dynein-dynactin-BICD2","machine":"dynein","label":"Key number: Stall force, human dynein-dynactin-BICD2","section":"summary"},{"source":"ref:belyy2016","machine":"dynein","label":"Belyy et al.","section":"sources","anchor":"ref-belyy2016","href":"https://doi.org/10.1038/ncb3393"}]},{"id":"dynein-tug-of-war","machine":"dynein","kind":"qa","prompt":"One dynein–dynactin–BICD2 complex was linked to one kinesin-1. How did the pair move?","answer":"The pairs crawled (median 26 nm/s), and about one in five moved toward the minus end.","explanation":"Without dynactin and BICD2N the pairs ran almost as fast as kinesin alone. One activated dynein can hold its own against one kinesin.","section":"evidence","topic":"numbers","sources":["evidence:belyy2016-tug-of-war","frontier:Balanced bidirectional transport","ref:belyy2016"],"tags":["kinesin","force"],"difficulty":2,"url":"/machines/dynein#evidence","cites":[{"source":"evidence:belyy2016-tug-of-war","machine":"dynein","label":"One dynein-dynactin-BICD2N complex against one kinesin-1 (Belyy V 2016)","section":"evidence","anchor":"ev-belyy2016-tug-of-war"},{"source":"frontier:Balanced bidirectional transport","machine":"dynein","label":"Open question: Balanced bidirectional transport","section":"summary"},{"source":"ref:belyy2016","machine":"dynein","label":"Belyy et al.","section":"sources","anchor":"ref-belyy2016","href":"https://doi.org/10.1038/ncb3393"}]},{"id":"dynein-two-dyneins","machine":"dynein","kind":"qa","prompt":"Why do dynein complexes built with the adaptors BICDR1 or HOOK3 pull harder and move faster than those built with BICD2?","answer":"They mostly recruit two dyneins per dynactin.","explanation":"Complexes with one active dynein and one tail that cannot walk were as slow as BICD2 complexes. With BICDR1 the stall force (6.5 pN) beat kinesin-1 (5.7 pN).","section":"evidence","topic":"numbers","sources":["evidence:urnavicius2018-two-dynein-force","evidence:urnavicius2018-two-dynein-speed","ref:urnavicius2018"],"tags":["dynactin","force","speed"],"difficulty":2,"url":"/machines/dynein#evidence","cites":[{"source":"evidence:urnavicius2018-two-dynein-force","machine":"dynein","label":"Stall force with one or two dyneins per dynactin (Urnavicius L 2018)","section":"evidence","anchor":"ev-urnavicius2018-two-dynein-force"},{"source":"evidence:urnavicius2018-two-dynein-speed","machine":"dynein","label":"Speed with one or two dyneins per dynactin (Urnavicius L 2018)","section":"evidence","anchor":"ev-urnavicius2018-two-dynein-speed"},{"source":"ref:urnavicius2018","machine":"dynein","label":"Urnavicius et al.","section":"sources","anchor":"ref-urnavicius2018","href":"https://doi.org/10.1038/nature25462"}]},{"id":"ribosome-purpose","machine":"ribosome","kind":"qa","prompt":"What does the ribosome do in a cell?","answer":"It reads an mRNA and builds the matching protein chain.","explanation":"It turns a nucleic acid sequence into a protein sequence. Every protein in your body comes off a ribosome.","section":"summary","topic":"purpose","sources":["machine:tagline","machine:summary","story:summary"],"tags":["translation"],"difficulty":1,"url":"/machines/ribosome#summary","cites":[{"source":"machine:tagline","machine":"ribosome","label":"Summary","section":"summary"},{"source":"machine:summary","machine":"ribosome","label":"Summary","section":"summary"},{"source":"story:summary","machine":"ribosome","label":"Big picture","section":"story"}]},{"id":"ribosome-small-subunit-role","machine":"ribosome","kind":"cloze","prompt":"The ribosome's {{small subunit}} checks each codon against the anticodon of the incoming tRNA.","answer":"small subunit","explanation":"In bacteria its core, 16S rRNA, reads the codon and holds the mRNA channel. The large subunit joins the amino acids.","section":"summary","topic":"parts","sources":["machine:summary","component:16S rRNA"],"tags":["decoding","subunits"],"difficulty":1,"url":"/machines/ribosome#summary","cites":[{"source":"machine:summary","machine":"ribosome","label":"Summary","section":"summary"},{"source":"component:16S rRNA","machine":"ribosome","label":"Part: 16S rRNA","section":"summary"}]},{"id":"ribosome-rna-catalyst","machine":"ribosome","kind":"cloze","prompt":"In the bacterial ribosome, the peptide bond is made by {{23S rRNA}} of the large subunit, not by protein.","answer":"23S rRNA","explanation":"Only 23S rRNA lines the catalytic centre, so the ribosome is a ribozyme: an enzyme made of RNA.","section":"summary","topic":"parts","sources":["component:23S rRNA","mechanism:Peptide bond formation","ref:nissen2000"],"tags":["ribozyme","rrna"],"difficulty":1,"url":"/machines/ribosome#summary","cites":[{"source":"component:23S rRNA","machine":"ribosome","label":"Part: 23S rRNA","section":"summary"},{"source":"mechanism:Peptide bond formation","machine":"ribosome","label":"Step: Peptide bond formation","section":"mechanism"},{"source":"ref:nissen2000","machine":"ribosome","label":"Nissen et al.","section":"sources","anchor":"ref-nissen2000","href":"https://doi.org/10.1126/science.289.5481.920"}]},{"id":"ribosome-protein-role","machine":"ribosome","kind":"qa","prompt":"No ribosomal protein sits in the catalytic centre. What job do the ribosomal proteins do instead?","answer":"They sit on the surface and stabilise the rRNA folds.","explanation":"The catalytic centre is all RNA, with proteins on the outside. That fits an RNA-first origin of the ribosome.","section":"summary","topic":"parts","sources":["evolution:RNA came first","component:30S proteins S2-S21","component:50S proteins L2-L36","ref:nissen2000","ref:ban2000"],"tags":["evolution","rrna"],"difficulty":2,"url":"/machines/ribosome#summary","cites":[{"source":"evolution:RNA came first","machine":"ribosome","label":"RNA came first","section":"summary"},{"source":"component:30S proteins S2-S21","machine":"ribosome","label":"Part: 30S proteins S2-S21","section":"summary"},{"source":"component:50S proteins L2-L36","machine":"ribosome","label":"Part: 50S proteins L2-L36","section":"summary"},{"source":"ref:nissen2000","machine":"ribosome","label":"Nissen et al.","section":"sources","anchor":"ref-nissen2000","href":"https://doi.org/10.1126/science.289.5481.920"},{"source":"ref:ban2000","machine":"ribosome","label":"Ban et al.","section":"sources","anchor":"ref-ban2000","href":"https://doi.org/10.1126/science.289.5481.905"}]},{"id":"ribosome-three-sites","machine":"ribosome","kind":"cloze","prompt":"A tRNA passes through the ribosome's three binding sites in the order {{A, P, E}}.","answer":"A, P, E","explanation":"The A site takes in the new aminoacyl-tRNA, the P site holds the tRNA that carries the chain, and the E site holds the empty tRNA until it leaves.","section":"summary","topic":"parts","sources":["stat:tRNA binding sites","step:The A site is open","ref:rheinberger1981"],"tags":["trna","sites"],"difficulty":1,"url":"/machines/ribosome#summary","cites":[{"source":"stat:tRNA binding sites","machine":"ribosome","label":"Key number: tRNA binding sites","section":"summary"},{"source":"step:The A site is open","machine":"ribosome","label":"Step: The A site is open","section":"mechanism"},{"source":"ref:rheinberger1981","machine":"ribosome","label":"Rheinberger, Sternbach and Nierhaus, PNAS 1981","section":"sources","anchor":"ref-rheinberger1981","href":"https://doi.org/10.1073/pnas.78.9.5310"}]},{"id":"ribosome-ecoli-rate","machine":"ribosome","kind":"qa","prompt":"How many amino acids per second does a ribosome add in E. coli, from slow to fast growth?","answer":"About 12 to 17.","explanation":"12 at slow growth and 17 at fast growth, measured by pulse labelling. At 17 per second, one full cycle takes about 60 ms.","section":"summary","topic":"numbers","sources":["stat:Elongation rate in E. coli","step:The A site is open","ref:young1976"],"tags":["speed"],"difficulty":2,"url":"/machines/ribosome#summary","cites":[{"source":"stat:Elongation rate in E. coli","machine":"ribosome","label":"Key number: Elongation rate in E. coli","section":"summary"},{"source":"step:The A site is open","machine":"ribosome","label":"Step: The A site is open","section":"mechanism"},{"source":"ref:young1976","machine":"ribosome","label":"Young and Bremer, Biochemical Journal 1976","section":"sources","anchor":"ref-young1976","href":"https://doi.org/10.1042/bj1600185"}]},{"id":"ribosome-error-rate","machine":"ribosome","kind":"cloze","prompt":"The ribosome's error rate is about {{10^-3 to 10^-5}} per elongation step.","answer":"10^-3 to 10^-5","explanation":"It holds this accuracy over thousands of cycles. The range is quoted from a review; measurements in E. coli span about 10^-6 to 10^-3 per codon.","section":"summary","topic":"numbers","sources":["stat:Error rate per step","evidence:manickam2014-misreading-floor","ref:rundlet2021"],"tags":["accuracy"],"difficulty":2,"url":"/machines/ribosome#summary","cites":[{"source":"stat:Error rate per step","machine":"ribosome","label":"Key number: Error rate per step","section":"summary"},{"source":"evidence:manickam2014-misreading-floor","machine":"ribosome","label":"Lowest missense error frequencies measured in vivo (Manickam N 2014)","section":"evidence","anchor":"ev-manickam2014-misreading-floor"},{"source":"ref:rundlet2021","machine":"ribosome","label":"Rundlet et al.","section":"sources","anchor":"ref-rundlet2021","href":"https://doi.org/10.1038/s41586-021-03713-x"}]},{"id":"ribosome-ribo-t","machine":"ribosome","kind":"qa","prompt":"What did tethering the two ribosomal subunits into one molecule (Ribo-T) show?","answer":"The subunits need not exchange: Ribo-T keeps E. coli alive without wild-type ribosomes.","explanation":"Short RNA linkers join small- and large-subunit rRNA. The tethered ribosome still makes protein, both in vitro and in cells.","section":"summary","topic":"debate","sources":["frontier:Tethered subunits","ref:orelle2015"],"tags":["engineering","subunits"],"difficulty":2,"url":"/machines/ribosome#summary","cites":[{"source":"frontier:Tethered subunits","machine":"ribosome","label":"Open question: Tethered subunits","section":"summary"},{"source":"ref:orelle2015","machine":"ribosome","label":"Orelle et al.","section":"sources","anchor":"ref-orelle2015","href":"https://doi.org/10.1038/nature14862"}]},{"id":"ribosome-non-natural-polymers","machine":"ribosome","kind":"qa","prompt":"What is the status of using ribosomes to make polymers that are not proteins?","answer":"Proposed: no such polymer chemistry yet runs on a ribosome at scale.","explanation":"The proposed starting point is a dedicated ribosome that is tethered and orthogonal (it reads only its own mRNA). Those parts, and ribosomes that read four-base codons, are each already demonstrated.","section":"summary","topic":"debate","sources":["frontier:Ribosomes for non-natural polymers","frontier:Tethered subunits","frontier:Private translation channels","frontier:Expanded genetic codes","ref:orelle2015","ref:rackham2005","ref:neumann2010"],"tags":["engineering","frontier"],"difficulty":2,"url":"/machines/ribosome#summary","cites":[{"source":"frontier:Ribosomes for non-natural polymers","machine":"ribosome","label":"Open question: Ribosomes for non-natural polymers","section":"summary"},{"source":"frontier:Tethered subunits","machine":"ribosome","label":"Open question: Tethered subunits","section":"summary"},{"source":"frontier:Private translation channels","machine":"ribosome","label":"Open question: Private translation channels","section":"summary"},{"source":"frontier:Expanded genetic codes","machine":"ribosome","label":"Open question: Expanded genetic codes","section":"summary"},{"source":"ref:orelle2015","machine":"ribosome","label":"Orelle et al.","section":"sources","anchor":"ref-orelle2015","href":"https://doi.org/10.1038/nature14862"},{"source":"ref:rackham2005","machine":"ribosome","label":"Rackham and Chin, Nature Chemical Biology 2005","section":"sources","anchor":"ref-rackham2005","href":"https://doi.org/10.1038/nchembio719"},{"source":"ref:neumann2010","machine":"ribosome","label":"Neumann et al.","section":"sources","anchor":"ref-neumann2010","href":"https://doi.org/10.1038/nature08817"}]},{"id":"ribosome-eftu-holds-end","machine":"ribosome","kind":"qa","prompt":"Why does EF-Tu hold the amino-acid end of an incoming tRNA while the anticodon reaches down to the codon?","answer":"To keep the amino acid away from the catalytic centre until the codon has been checked.","explanation":"This is the A/T state. EF-Tu complexes also bind and fall off fast, so the ribosome can try and reject many wrong tRNAs at little cost.","section":"mechanism","topic":"cycle","sources":["step:EF-Tu brings an aminoacyl-tRNA","mechanism:Delivery","ref:pape1998"],"tags":["ef-tu","decoding"],"difficulty":2,"url":"/machines/ribosome#mechanism","cites":[{"source":"step:EF-Tu brings an aminoacyl-tRNA","machine":"ribosome","label":"Step: EF-Tu brings an aminoacyl-tRNA","section":"mechanism"},{"source":"mechanism:Delivery","machine":"ribosome","label":"Step: Delivery","section":"mechanism"},{"source":"ref:pape1998","machine":"ribosome","label":"Pape, Wintermeyer and Rodnina, EMBO J 1998","section":"sources","href":"https://doi.org/10.1093/emboj/17.24.7490"}]},{"id":"ribosome-decoding-trigger","machine":"ribosome","kind":"qa","prompt":"On the ribosome, what does a correct codon–anticodon pair switch on?","answer":"GTP hydrolysis by EF-Tu, which then releases the tRNA into the A site.","explanation":"16S rRNA bases, among them A1492 and A1493, recognise the correct pair, and the small subunit closes around the tRNA. The GTPase rate differs most between right and wrong tRNAs, so it largely decides which tRNA is accepted.","section":"mechanism","topic":"cycle","sources":["step:The codon is checked","mechanism:Decoding","ref:voorhees2010","ref:pape1998"],"tags":["decoding","ef-tu","gtp"],"difficulty":2,"url":"/machines/ribosome#mechanism","cites":[{"source":"step:The codon is checked","machine":"ribosome","label":"Step: The codon is checked","section":"mechanism"},{"source":"mechanism:Decoding","machine":"ribosome","label":"Step: Decoding","section":"mechanism"},{"source":"ref:voorhees2010","machine":"ribosome","label":"Voorhees et al.","section":"sources","anchor":"ref-voorhees2010","href":"https://doi.org/10.1126/science.1194460"},{"source":"ref:pape1998","machine":"ribosome","label":"Pape, Wintermeyer and Rodnina, EMBO J 1998","section":"sources","href":"https://doi.org/10.1093/emboj/17.24.7490"}]},{"id":"ribosome-hybrid-states","machine":"ribosome","kind":"cloze","prompt":"After the ribosome forms a peptide bond, the tRNA {{acceptor ends}} move on the large subunit while the anticodons stay put, giving hybrid A/P and P/E states.","answer":"acceptor ends","explanation":"Translocation happens in two moves, so the acceptor ends and then the anticodons shift one at a time.","section":"mechanism","topic":"cycle","sources":["mechanism:Hybrid states","step:The subunits ratchet","evidence:moazed1989-hybrid-states","ref:moazed1989"],"tags":["translocation","trna"],"difficulty":2,"url":"/machines/ribosome#mechanism","cites":[{"source":"mechanism:Hybrid states","machine":"ribosome","label":"Step: Hybrid states","section":"mechanism"},{"source":"step:The subunits ratchet","machine":"ribosome","label":"Step: The subunits ratchet","section":"mechanism"},{"source":"evidence:moazed1989-hybrid-states","machine":"ribosome","label":"Number of discrete steps in tRNA translocation (Moazed D 1989)","section":"evidence","anchor":"ev-moazed1989-hybrid-states"},{"source":"ref:moazed1989","machine":"ribosome","label":"Moazed and Noller, Nature 1989","section":"sources","anchor":"ref-moazed1989","href":"https://doi.org/10.1038/342142a0"}]},{"id":"ribosome-ratchet-driver","machine":"ribosome","kind":"qa","prompt":"What carries the ribosome's small subunit into its rotated (ratcheted) state after the peptide bond forms?","answer":"Thermal motion: the move happens on its own, without EF-G or GTP.","explanation":"The small subunit turns about 10° against the large subunit. The move is spontaneous and reversible.","section":"mechanism","topic":"cycle","sources":["step:The subunits ratchet","ref:rundlet2021"],"tags":["translocation","rotation"],"difficulty":3,"url":"/machines/ribosome#mechanism","cites":[{"source":"step:The subunits ratchet","machine":"ribosome","label":"Step: The subunits ratchet","section":"mechanism"},{"source":"ref:rundlet2021","machine":"ribosome","label":"Rundlet et al.","section":"sources","anchor":"ref-rundlet2021","href":"https://doi.org/10.1038/s41586-021-03713-x"}]},{"id":"ribosome-efg-catches","machine":"ribosome","kind":"qa","prompt":"What does EF-G·GTP do when it first binds the ribosome after the subunits have rotated?","answer":"It catches and holds the rotated state, which the ribosome reaches on its own, and starts to unlock the small subunit.","explanation":"In the structures of Rundlet and colleagues, EF-G still carries unsplit GTP at this point. So the energy of GTP is spent later in translocation than once thought.","section":"mechanism","topic":"cycle","sources":["step:EF-G binds with GTP","mechanism:Ratchet and translocation","ref:rundlet2021"],"tags":["ef-g","gtp","translocation"],"difficulty":3,"url":"/machines/ribosome#mechanism","cites":[{"source":"step:EF-G binds with GTP","machine":"ribosome","label":"Step: EF-G binds with GTP","section":"mechanism"},{"source":"mechanism:Ratchet and translocation","machine":"ribosome","label":"Step: Ratchet and translocation","section":"mechanism"},{"source":"ref:rundlet2021","machine":"ribosome","label":"Rundlet et al.","section":"sources","anchor":"ref-rundlet2021","href":"https://doi.org/10.1038/s41586-021-03713-x"}]},{"id":"ribosome-reading-frame","machine":"ribosome","kind":"qa","prompt":"Why must the ribosome move the mRNA by exactly three nucleotides in each cycle?","answer":"To keep the reading frame.","explanation":"EF-G splits its GTP and the small-subunit head swivels, and the tRNA anticodons move with the mRNA by one codon. A kink in the mRNA, held by a metal ion, may also stop it from slipping out of frame.","section":"mechanism","topic":"cycle","sources":["step:EF-G moves the tRNAs one codon","step:EF-G leaves","ref:wen2008","ref:selmer2006"],"tags":["translocation","mrna"],"difficulty":1,"url":"/machines/ribosome#mechanism","cites":[{"source":"step:EF-G moves the tRNAs one codon","machine":"ribosome","label":"Step: EF-G moves the tRNAs one codon","section":"mechanism"},{"source":"step:EF-G leaves","machine":"ribosome","label":"Step: EF-G leaves","section":"mechanism"},{"source":"ref:wen2008","machine":"ribosome","label":"Wen et al.","section":"sources","href":"https://doi.org/10.1038/nature06716"},{"source":"ref:selmer2006","machine":"ribosome","label":"Selmer et al.","section":"sources","anchor":"ref-selmer2006","href":"https://doi.org/10.1126/science.1131127"}]},{"id":"ribosome-exit-tunnel","machine":"ribosome","kind":"qa","prompt":"Why can't a new protein chain fold into domains inside the ribosome's exit tunnel?","answer":"The tunnel is too narrow for any fold larger than an alpha-helix.","explanation":"The chain runs from the catalytic centre through the large subunit to the surface. Chains that cannot fold alone are then caught by GroEL.","section":"mechanism","topic":"cycle","sources":["mechanism:The chain leaves through the tunnel","evidence:voss2006-tunnel-width","link:groel","ref:voss2006"],"tags":["tunnel","folding"],"difficulty":2,"url":"/machines/ribosome#mechanism","cites":[{"source":"mechanism:The chain leaves through the tunnel","machine":"ribosome","label":"Step: The chain leaves through the tunnel","section":"mechanism"},{"source":"evidence:voss2006-tunnel-width","machine":"ribosome","label":"Width of the polypeptide exit tunnel (Voss NR 2006)","section":"evidence","anchor":"ev-voss2006-tunnel-width"},{"source":"link:groel","machine":"ribosome","label":"Link to GroEL–GroES","section":"story"},{"source":"ref:voss2006","machine":"ribosome","label":"Voss et al.","section":"sources","anchor":"ref-voss2006","href":"https://doi.org/10.1016/j.jmb.2006.05.023"}]},{"id":"ribosome-human-cell-count","machine":"ribosome","kind":"cloze","prompt":"One human HeLa cell holds about {{3.3–9.5 million}} ribosomes.","answer":"3.3–9.5 million","explanation":"3.3 million comes from the mass of 28S rRNA, 9.5 million from the total RNA per cell. Ribosomes make up about 6% of a HeLa cell's protein mass.","section":"story","topic":"numbers","sources":["fact:R4","stop:cell","fact:R6","ref:bnid107552","ref:bnid107347"],"tags":["scale","copies"],"difficulty":2,"url":"/machines/ribosome#story","cites":[{"source":"fact:R4","machine":"ribosome","label":"Ribosomes in one human cell (HeLa): 3.3–9.5 × 10^6","section":"story"},{"source":"stop:cell","machine":"ribosome","label":"Big picture: One cell","section":"story"},{"source":"fact:R6","machine":"ribosome","label":"Share of the protein mass of a HeLa cell that is ribosomes: 6 %","section":"story"},{"source":"ref:bnid107552","machine":"ribosome","label":"BNID 107552","section":"sources","href":"https://bionumbers.hms.harvard.edu/bionumber.aspx?id=107552"},{"source":"ref:bnid107347","machine":"ribosome","label":"BNID 107347","section":"sources","href":"https://bionumbers.hms.harvard.edu/bionumber.aspx?id=107347"}]},{"id":"ribosome-body-protein","machine":"ribosome","kind":"qa","prompt":"About how much protein does a human body build each day?","answer":"A few hundred grams (estimates run from about 175 to 400 g).","explanation":"All of it comes off ribosomes. Protein turnover takes about 20% of resting energy in an average healthy young adult.","section":"story","topic":"purpose","sources":["fact:R2","fact:R1","fact:R3","stop:body","ref:schutz2011","ref:norton1981","ref:welle1990"],"tags":["scale","body"],"difficulty":2,"url":"/machines/ribosome#story","cites":[{"source":"fact:R2","machine":"ribosome","label":"Protein turnover in the whole body: 300–400 g per day","section":"story"},{"source":"fact:R1","machine":"ribosome","label":"Protein made by the whole body per day, small study: 175 g per day","section":"story"},{"source":"fact:R3","machine":"ribosome","label":"Share of resting energy spent on protein turnover: 20 %","section":"story"},{"source":"stop:body","machine":"ribosome","label":"Big picture: You, building","section":"story"},{"source":"ref:schutz2011","machine":"ribosome","label":"Schutz 2011","section":"sources","href":"https://doi.org/10.1024/0300-9831/a000064"},{"source":"ref:norton1981","machine":"ribosome","label":"Norton 1981","section":"sources","href":"https://doi.org/10.1097/00000658-198108000-00001"},{"source":"ref:welle1990","machine":"ribosome","label":"Welle 1990","section":"sources","href":"https://doi.org/10.1152/ajpendo.1990.258.6.e990"}]},{"id":"ribosome-titin-time","machine":"ribosome","kind":"qa","prompt":"At the mouse stem-cell rate of 5.6 amino acids per second, about how long would one ribosome take to build titin (34,350 amino acids)?","answer":"About 1.7 hours.","explanation":"34,350 ÷ 5.6 ≈ 6,130 s. A typical 400-residue protein takes about 71 s. Both are calculations, not measured times.","section":"story","topic":"numbers","sources":["fact:R11","fact:R9","ref:uniprot-titin","ref:ingolia2011"],"tags":["speed","scale"],"difficulty":2,"url":"/machines/ribosome#story","cites":[{"source":"fact:R11","machine":"ribosome","label":"Time to build a typical protein: 71 s","section":"story"},{"source":"fact:R9","machine":"ribosome","label":"Speed of a ribosome in mammal cells: 5.6 amino acids per second","section":"story"},{"source":"ref:uniprot-titin","machine":"ribosome","label":"UniProt Q8WZ42","section":"sources","href":"https://rest.uniprot.org/uniprotkb/Q8WZ42"},{"source":"ref:ingolia2011","machine":"ribosome","label":"Ingolia 2011","section":"sources","href":"https://doi.org/10.1016/j.cell.2011.10.002"}]},{"id":"ribosome-printer-analogy","machine":"ribosome","kind":"qa","prompt":"Where does the analogy of the ribosome as 'a 3D printer that reads a tape' break down?","answer":"The ribosome does not place units by position: tRNA adapters bring each one, and it only checks that the adapter pairs with the code.","explanation":"The chain also folds itself into shape afterwards. What the analogy gets right: it reads a coded tape and builds a chain one unit at a time.","section":"story","topic":"purpose","sources":["analogy:A 3D printer that reads a tape"],"tags":["analogy","trna"],"difficulty":2,"url":"/machines/ribosome#story","cites":[{"source":"analogy:A 3D printer that reads a tape","machine":"ribosome","label":"Analogy: A 3D printer that reads a tape","section":"story"}]},{"id":"ribosome-factory-line-analogy","machine":"ribosome","kind":"qa","prompt":"Many ribosomes read one mRNA at once. Why is 'a factory line' a misleading picture of this?","answer":"All the ribosomes on the message make the same product: they are copies, not stations with different jobs.","explanation":"Each ribosome further along the message carries a longer chain. The first such ribosome clusters ever seen were making hemoglobin.","section":"story","topic":"purpose","sources":["analogy:A factory line","stop:polysome","fact:R7","link:hemoglobin","ref:warner1962"],"tags":["analogy","polysome"],"difficulty":2,"url":"/machines/ribosome#story","cites":[{"source":"analogy:A factory line","machine":"ribosome","label":"Analogy: A factory line","section":"story"},{"source":"stop:polysome","machine":"ribosome","label":"Big picture: String of ribosomes","section":"story"},{"source":"fact:R7","machine":"ribosome","label":"Protein made by the first ribosome clusters ever seen: Hemoglobin","section":"story"},{"source":"link:hemoglobin","machine":"ribosome","label":"Link to Hemoglobin","section":"story"},{"source":"ref:warner1962","machine":"ribosome","label":"Warner 1962","section":"sources","href":"https://doi.org/10.1126/science.138.3548.1399"}]},{"id":"ribosome-proteasome-balance","machine":"ribosome","kind":"qa","prompt":"In mouse L929 cells, how does the number of proteins the proteasome destroys per minute compare with the number ribosomes make?","answer":"About half as many.","explanation":"The ribosomes in one such cell make about 4 million proteins a minute. The proteasome destroys what the ribosome makes, which sets each protein's lifetime.","section":"story","topic":"numbers","sources":["link:proteasome","fact:R5","ref:princiotta2003"],"tags":["proteasome","turnover"],"difficulty":3,"url":"/machines/ribosome#story","cites":[{"source":"link:proteasome","machine":"ribosome","label":"Link to Proteasome","section":"story"},{"source":"fact:R5","machine":"ribosome","label":"Proteins made per minute in one mouse cell: 4 × 10^6 per minute","section":"story"},{"source":"ref:princiotta2003","machine":"ribosome","label":"Princiotta 2003","section":"sources","href":"https://doi.org/10.1016/s1074-7613(03)00051-7"}]},{"id":"ribosome-young-method","machine":"ribosome","kind":"qa","prompt":"How did Young and Bremer (1976) measure the ribosome's elongation rate in E. coli?","answer":"By pulse labelling: they timed how long proteins of each size took to become fully labelled with a pulse of radioactive leucine.","explanation":"The delay grows with protein size, and the slope gives the chain speed: 17 amino acids per second at fast growth and 12 at slow growth, at 37 °C.","section":"evidence","topic":"numbers","sources":["evidence:young1976-elongation-rate","ref:young1976"],"tags":["speed","method"],"difficulty":2,"url":"/machines/ribosome#evidence","cites":[{"source":"evidence:young1976-elongation-rate","machine":"ribosome","label":"Peptide-chain elongation rate in E. coli at three growth rates (Young R 1976)","section":"evidence","anchor":"ev-young1976-elongation-rate"},{"source":"ref:young1976","machine":"ribosome","label":"Young and Bremer, Biochemical Journal 1976","section":"sources","anchor":"ref-young1976","href":"https://doi.org/10.1042/bj1600185"}]},{"id":"ribosome-ingolia-method","machine":"ribosome","kind":"qa","prompt":"How did Ingolia and colleagues (2011) measure the elongation rate in mouse embryonic stem cells?","answer":"They blocked new initiation with harringtonine, then used ribosome profiling to track how fast the front of the ribosome-free zone moved along genes.","explanation":"The front moved at 5.6 ± 0.5 codons per second, averaged over thousands of genes: slower than the 12–17 per second of E. coli.","section":"evidence","topic":"numbers","sources":["evidence:ingolia2011-mammalian-elongation","ref:ingolia2011"],"tags":["speed","method"],"difficulty":3,"url":"/machines/ribosome#evidence","cites":[{"source":"evidence:ingolia2011-mammalian-elongation","machine":"ribosome","label":"Translation elongation rate in mouse embryonic stem cells (Ingolia NT 2011)","section":"evidence","anchor":"ev-ingolia2011-mammalian-elongation"},{"source":"ref:ingolia2011","machine":"ribosome","label":"Ingolia 2011","section":"sources","href":"https://doi.org/10.1016/j.cell.2011.10.002"}]},{"id":"ribosome-wen-step","machine":"ribosome","kind":"cloze","prompt":"Held in optical tweezers, a single E. coli ribosome advanced along an mRNA hairpin by {{about 3 nucleotides}} per step.","answer":"about 3 nucleotides","explanation":"The measured step was 2.94 ± 0.72 nucleotides, one codon, taken in about 0.078 s. The pauses between steps (median 2.8 s) took far longer than the steps.","section":"evidence","topic":"numbers","sources":["evidence:wen2008-codon-step","step:EF-G moves the tRNAs one codon","ref:wen2008"],"tags":["translocation","single-molecule"],"difficulty":2,"url":"/machines/ribosome#evidence","cites":[{"source":"evidence:wen2008-codon-step","machine":"ribosome","label":"Translocation step of a single ribosome (Wen JD 2008)","section":"evidence","anchor":"ev-wen2008-codon-step"},{"source":"step:EF-G moves the tRNAs one codon","machine":"ribosome","label":"Step: EF-G moves the tRNAs one codon","section":"mechanism"},{"source":"ref:wen2008","machine":"ribosome","label":"Wen et al.","section":"sources","href":"https://doi.org/10.1038/nature06716"}]},{"id":"ribosome-pape-slow-steps","machine":"ribosome","kind":"qa","prompt":"In Pape and colleagues' (1998) kinetics of tRNA selection on E. coli ribosomes, which two steps were slowest?","answer":"Accommodation (the tRNA swinging into the A site, about 8 per s) and EF-Tu·GDP release (about 4 per s).","explanation":"Codon reading (about 100 per s) and GTPase activation (about 500 per s) are fast. Measured at 20 °C; the peptide bond does not wait for EF-Tu·GDP to leave.","section":"evidence","topic":"numbers","sources":["evidence:pape1998-decoding-kinetics","step:The tRNA swings into the A site","ref:pape1998"],"tags":["kinetics","ef-tu"],"difficulty":3,"url":"/machines/ribosome#evidence","cites":[{"source":"evidence:pape1998-decoding-kinetics","machine":"ribosome","label":"Rate constants of EF-Tu-dependent aminoacyl-tRNA binding to the A site (Pape T 1998)","section":"evidence","anchor":"ev-pape1998-decoding-kinetics"},{"source":"step:The tRNA swings into the A site","machine":"ribosome","label":"Step: The tRNA swings into the A site","section":"mechanism"},{"source":"ref:pape1998","machine":"ribosome","label":"Pape, Wintermeyer and Rodnina, EMBO J 1998","section":"sources","href":"https://doi.org/10.1093/emboj/17.24.7490"}]},{"id":"ribosome-nissen-distance","machine":"ribosome","kind":"cloze","prompt":"In the Haloarcula 50S crystal structure with substrate analogues bound, no protein side-chain atom came closer than {{about 18 Å}} to the forming peptide bond.","answer":"about 18 Å","explanation":"Only conserved 23S rRNA touched the substrates, so the catalyst is RNA: the ribosome is a ribozyme.","section":"evidence","topic":"numbers","sources":["evidence:nissen2000-rna-catalyst","stat:Protein near the new peptide bond","ref:nissen2000"],"tags":["ribozyme","structure"],"difficulty":2,"url":"/machines/ribosome#evidence","cites":[{"source":"evidence:nissen2000-rna-catalyst","machine":"ribosome","label":"Distance from the forming peptide bond to the nearest protein (Nissen P 2000)","section":"evidence","anchor":"ev-nissen2000-rna-catalyst"},{"source":"stat:Protein near the new peptide bond","machine":"ribosome","label":"Key number: Protein near the new peptide bond","section":"summary"},{"source":"ref:nissen2000","machine":"ribosome","label":"Nissen et al.","section":"sources","anchor":"ref-nissen2000","href":"https://doi.org/10.1126/science.289.5481.920"}]},{"id":"ribosome-error-floor","machine":"ribosome","kind":"qa","prompt":"Using a sensitive β-galactosidase reporter in E. coli, how low were most missense error rates that Manickam et al. (2014) measured?","answer":"About 2.3 × 10^-6 per codon (10 of 14 codons tested).","explanation":"Four error-prone codons were about 100-fold higher. With Kramer and Farabaugh 2007, per-codon errors span about 10^-6 to 10^-3, while the page range of 10^-3 to 10^-5 is quoted from a review.","section":"evidence","topic":"debate","sources":["evidence:manickam2014-misreading-floor","evidence:kramer2007-missense-rates","stat:Error rate per step","ref:manickam2014"],"tags":["accuracy","contested"],"difficulty":3,"url":"/machines/ribosome#evidence","cites":[{"source":"evidence:manickam2014-misreading-floor","machine":"ribosome","label":"Lowest missense error frequencies measured in vivo (Manickam N 2014)","section":"evidence","anchor":"ev-manickam2014-misreading-floor"},{"source":"evidence:kramer2007-missense-rates","machine":"ribosome","label":"Missense error frequency of tRNALys at near-cognate codons in vivo (Kramer EB 2007)","section":"evidence","anchor":"ev-kramer2007-missense-rates"},{"source":"stat:Error rate per step","machine":"ribosome","label":"Key number: Error rate per step","section":"summary"},{"source":"ref:manickam2014","machine":"ribosome","label":"Manickam et al.","section":"sources","href":"https://doi.org/10.1261/rna.039792.113"}]},{"id":"rna-polymerase-makes-mrna","machine":"rna-polymerase","kind":"cloze","prompt":"RNA polymerase II copies a DNA gene into {{messenger RNA}}, the molecule that ribosomes read.","answer":"messenger RNA","explanation":"It works one base at a time: it pairs the template strand with a growing RNA chain.","section":"summary","topic":"purpose","sources":["machine:summary","machine:tagline"],"tags":["purpose","mrna"],"difficulty":1,"url":"/machines/rna-polymerase#summary","cites":[{"source":"machine:summary","machine":"rna-polymerase","label":"Summary","section":"summary"},{"source":"machine:tagline","machine":"rna-polymerase","label":"Summary","section":"summary"}]},{"id":"rna-polymerase-energy-source","machine":"rna-polymerase","kind":"qa","prompt":"What pays for each nucleotide that RNA polymerase II adds to the RNA?","answer":"The incoming nucleoside triphosphate itself: the energy of its triphosphate.","explanation":"The new bond takes the energy of the triphosphate, and pyrophosphate is split off and leaves.","section":"summary","topic":"purpose","sources":["machine:energy","step:The bond forms"],"tags":["energy","ntp"],"difficulty":1,"url":"/machines/rna-polymerase#summary","cites":[{"source":"machine:energy","machine":"rna-polymerase","label":"Summary","section":"summary"},{"source":"step:The bond forms","machine":"rna-polymerase","label":"Step: The bond forms","section":"mechanism"}]},{"id":"rna-polymerase-cleft-subunits","machine":"rna-polymerase","kind":"cloze","prompt":"In RNA polymerase II, the two largest subunits, {{Rpb1 and Rpb2}}, build the DNA cleft and the active site.","answer":"Rpb1 and Rpb2","explanation":"Rpb1 also carries the bridge helix, the trigger loop, the clamp core and metal A; Rpb2 forms the second wall of the cleft.","section":"summary","topic":"parts","sources":["stat:Subunits","component:Rpb1","component:Rpb2","ref:bushnell2003"],"tags":["subunits","active-site"],"difficulty":2,"url":"/machines/rna-polymerase#summary","cites":[{"source":"stat:Subunits","machine":"rna-polymerase","label":"Key number: Subunits","section":"summary"},{"source":"component:Rpb1","machine":"rna-polymerase","label":"Part: Rpb1","section":"summary"},{"source":"component:Rpb2","machine":"rna-polymerase","label":"Part: Rpb2","section":"summary"},{"source":"ref:bushnell2003","machine":"rna-polymerase","label":"Bushnell and Kornberg, PNAS 2003","section":"sources","anchor":"ref-bushnell2003","href":"https://doi.org/10.1073/pnas.1130601100"}]},{"id":"rna-polymerase-rpb4-rpb7-role","machine":"rna-polymerase","kind":"qa","prompt":"Which part of RNA polymerase II sits next to the RNA exit groove and binds the emerging transcript?","answer":"The Rpb4-Rpb7 stalk.","explanation":"Crystal structures also show Rpb7 wedging between the clamp and the linker to the tail domain, which locks the clamp closed.","section":"summary","topic":"parts","sources":["component:Rpb4-Rpb7 stalk","evidence:reconstituted-pol2-twelve-subunits","evidence:complete-pol2-twelve-subunits"],"tags":["subunits","rna-exit"],"difficulty":2,"url":"/machines/rna-polymerase#summary","cites":[{"source":"component:Rpb4-Rpb7 stalk","machine":"rna-polymerase","label":"Part: Rpb4-Rpb7 stalk","section":"summary"},{"source":"evidence:reconstituted-pol2-twelve-subunits","machine":"rna-polymerase","label":"Subunit count of complete RNA polymerase II, independent structure (Armache KJ 2003)","section":"evidence","anchor":"ev-reconstituted-pol2-twelve-subunits"},{"source":"evidence:complete-pol2-twelve-subunits","machine":"rna-polymerase","label":"Subunit count of complete RNA polymerase II (Bushnell DA 2003)","section":"evidence","anchor":"ev-complete-pol2-twelve-subunits"}]},{"id":"rna-polymerase-subunit-count","machine":"rna-polymerase","kind":"cloze","prompt":"Complete RNA polymerase II has {{12}} subunits: a ten-subunit core plus the Rpb4-Rpb7 pair.","answer":"12","explanation":"Bushnell and Kornberg tagged Rpb4 so every purified enzyme carried the pair, then solved the whole complex at 4.1 Å.","section":"summary","topic":"numbers","sources":["stat:Subunits","evidence:complete-pol2-twelve-subunits","ref:bushnell2003"],"tags":["subunits"],"difficulty":1,"url":"/machines/rna-polymerase#summary","cites":[{"source":"stat:Subunits","machine":"rna-polymerase","label":"Key number: Subunits","section":"summary"},{"source":"evidence:complete-pol2-twelve-subunits","machine":"rna-polymerase","label":"Subunit count of complete RNA polymerase II (Bushnell DA 2003)","section":"evidence","anchor":"ev-complete-pol2-twelve-subunits"},{"source":"ref:bushnell2003","machine":"rna-polymerase","label":"Bushnell and Kornberg, PNAS 2003","section":"sources","anchor":"ref-bushnell2003","href":"https://doi.org/10.1073/pnas.1130601100"}]},{"id":"rna-polymerase-hybrid-length","machine":"rna-polymerase","kind":"cloze","prompt":"Inside transcribing RNA polymerase II, the DNA-RNA hybrid is {{9}} base pairs long.","answer":"9","explanation":"The hybrid runs from the active site at nearly a right angle to the entering DNA (yeast Pol II crystal structure, 3.3 Å).","section":"summary","topic":"numbers","sources":["stat:DNA-RNA hybrid length","evidence:dna-rna-hybrid-nine-bp","ref:gnatt2001"],"tags":["hybrid","structure"],"difficulty":1,"url":"/machines/rna-polymerase#summary","cites":[{"source":"stat:DNA-RNA hybrid length","machine":"rna-polymerase","label":"Key number: DNA-RNA hybrid length","section":"summary"},{"source":"evidence:dna-rna-hybrid-nine-bp","machine":"rna-polymerase","label":"Length of the DNA-RNA hybrid in the elongation complex (Gnatt AL 2001)","section":"evidence","anchor":"ev-dna-rna-hybrid-nine-bp"},{"source":"ref:gnatt2001","machine":"rna-polymerase","label":"Gnatt et al.","section":"sources","anchor":"ref-gnatt2001","href":"https://doi.org/10.1126/science.1059495"}]},{"id":"rna-polymerase-antibiotic-target","machine":"rna-polymerase","kind":"qa","prompt":"Which state of RNA polymerase is an explicit target for new antibiotics, according to structures with streptolydigin?","answer":"The preinsertion state: the NTP held before the trigger loop folds over it.","explanation":"Streptolydigin holds the trigger loop away, so the nucleotide stays in an inactive preinsertion position. This inhibitor-design idea is lab-scale.","section":"summary","topic":"debate","sources":["frontier:Structure-guided inhibitor design","evolution:Shared two-step substrate path","ref:vassylyev2007b"],"tags":["trigger-loop","drugs"],"difficulty":3,"url":"/machines/rna-polymerase#summary","cites":[{"source":"frontier:Structure-guided inhibitor design","machine":"rna-polymerase","label":"Open question: Structure-guided inhibitor design","section":"summary"},{"source":"evolution:Shared two-step substrate path","machine":"rna-polymerase","label":"Shared two-step substrate path","section":"summary"},{"source":"ref:vassylyev2007b","machine":"rna-polymerase","label":"Vassylyev et al.","section":"sources","anchor":"ref-vassylyev2007b","href":"https://doi.org/10.1038/nature05931"}]},{"id":"rna-polymerase-t7-private-channel","machine":"rna-polymerase","kind":"qa","prompt":"Why does phage T7 RNA polymerase give a private expression channel in an E. coli cell?","answer":"It transcribes only T7 promoters, so it copies only the chosen gene.","explanation":"One subunit does the whole job and needs no factors. It is still the standard tool for recombinant protein work.","section":"summary","topic":"debate","sources":["frontier:Orthogonal expression with T7 RNA polymerase","species:T7 RNA polymerase","ref:studier1986"],"tags":["t7","biotech"],"difficulty":2,"url":"/machines/rna-polymerase#summary","cites":[{"source":"frontier:Orthogonal expression with T7 RNA polymerase","machine":"rna-polymerase","label":"Open question: Orthogonal expression with T7 RNA polymerase","section":"summary"},{"source":"species:T7 RNA polymerase","machine":"rna-polymerase","label":"T7 RNA polymerase","section":"summary"},{"source":"ref:studier1986","machine":"rna-polymerase","label":"Studier and Moffatt, Journal of Molecular Biology 1986","section":"sources","anchor":"ref-studier1986","href":"https://doi.org/10.1016/0022-2836(86)90385-2"}]},{"id":"rna-polymerase-post-translocation","machine":"rna-polymerase","kind":"qa","prompt":"In which translocation state can an NTP pair in the addition site of RNA polymerase II?","answer":"The post-translocation state, with template base i+1 facing the empty addition site.","explanation":"Until an NTP binds and holds it forward, the enzyme can still slide back to the pre-translocation state by thermal motion.","section":"mechanism","topic":"cycle","sources":["step:The addition site is open"],"tags":["translocation","ntp"],"difficulty":2,"url":"/machines/rna-polymerase#mechanism","cites":[{"source":"step:The addition site is open","machine":"rna-polymerase","label":"Step: The addition site is open","section":"mechanism"}]},{"id":"rna-polymerase-trigger-loop-folds","machine":"rna-polymerase","kind":"qa","prompt":"What does the trigger loop of RNA polymerase II do when a matching NTP pairs in the addition site?","answer":"It folds into a hairpin under the NTP, touches its base, sugar and phosphates, and seals the active site.","explanation":"Leu1081 touches the base, Gln1078 reaches the 3′-OH through Asn479, and His1085 binds the β-phosphate.","section":"mechanism","topic":"cycle","sources":["step:The trigger loop folds","mechanism:Substrate selection","ref:wang2006"],"tags":["trigger-loop"],"difficulty":2,"url":"/machines/rna-polymerase#mechanism","cites":[{"source":"step:The trigger loop folds","machine":"rna-polymerase","label":"Step: The trigger loop folds","section":"mechanism"},{"source":"mechanism:Substrate selection","machine":"rna-polymerase","label":"Step: Substrate selection","section":"mechanism"},{"source":"ref:wang2006","machine":"rna-polymerase","label":"Wang et al.","section":"sources","anchor":"ref-wang2006","href":"https://doi.org/10.1016/j.cell.2006.11.023"}]},{"id":"rna-polymerase-trigger-loop-coupling","machine":"rna-polymerase","kind":"qa","prompt":"Why does the trigger loop link NTP recognition to catalysis in RNA polymerase II?","answer":"The same loop that checks the NTP places His1085 where it may trigger the reaction.","explanation":"The loop closes over a correct NTP, so recognising the NTP and catalysis are coupled.","section":"mechanism","topic":"cycle","sources":["step:The trigger loop folds","evidence:wang2006-trigger-loop-contacts","ref:wang2006"],"tags":["trigger-loop","fidelity"],"difficulty":3,"url":"/machines/rna-polymerase#mechanism","cites":[{"source":"step:The trigger loop folds","machine":"rna-polymerase","label":"Step: The trigger loop folds","section":"mechanism"},{"source":"evidence:wang2006-trigger-loop-contacts","machine":"rna-polymerase","label":"Trigger-loop contacts with the NTP in the addition site (Wang D 2006)","section":"evidence","anchor":"ev-wang2006-trigger-loop-contacts"},{"source":"ref:wang2006","machine":"rna-polymerase","label":"Wang et al.","section":"sources","anchor":"ref-wang2006","href":"https://doi.org/10.1016/j.cell.2006.11.023"}]},{"id":"rna-polymerase-two-metals-roles","machine":"rna-polymerase","kind":"qa","prompt":"Of the two Mg2+ ions in the active site of RNA polymerase II, which stays and which comes and goes each cycle?","answer":"Metal A stays bound to Rpb1 aspartates; metal B comes in with the NTP and leaves with the pyrophosphate.","explanation":"Metal A sits by the RNA 3′-OH and metal B holds the triphosphate, about 4 Å apart.","section":"mechanism","topic":"cycle","sources":["step:Pyrophosphate leaves","step:The NTP pairs with the template","stat:Active-site metals","step:The bond forms","evidence:metal-site-spacing","ref:wang2006"],"tags":["metals","catalysis"],"difficulty":2,"url":"/machines/rna-polymerase#mechanism","cites":[{"source":"step:Pyrophosphate leaves","machine":"rna-polymerase","label":"Step: Pyrophosphate leaves","section":"mechanism"},{"source":"step:The NTP pairs with the template","machine":"rna-polymerase","label":"Step: The NTP pairs with the template","section":"mechanism"},{"source":"stat:Active-site metals","machine":"rna-polymerase","label":"Key number: Active-site metals","section":"summary"},{"source":"step:The bond forms","machine":"rna-polymerase","label":"Step: The bond forms","section":"mechanism"},{"source":"evidence:metal-site-spacing","machine":"rna-polymerase","label":"Spacing of the Mg2+ sites in transcribing Pol II (Wang D 2006)","section":"evidence","anchor":"ev-metal-site-spacing"},{"source":"ref:wang2006","machine":"rna-polymerase","label":"Wang et al.","section":"sources","anchor":"ref-wang2006","href":"https://doi.org/10.1016/j.cell.2006.11.023"}]},{"id":"rna-polymerase-loop-opens-before-ppi","machine":"rna-polymerase","kind":"qa","prompt":"Why must the trigger loop of RNA polymerase II open before pyrophosphate can leave?","answer":"The closed loop blocks the way out and holds the pyrophosphate through His1085.","explanation":"Only the fully open loop lets pyrophosphate move back to the entry site and out through the pore (Yi et al. 2026 preprint; Li et al. 2026 agrees).","section":"mechanism","topic":"cycle","sources":["step:The trigger loop opens","evidence:yi2026-ppi-bound-closed-loop","evidence:li2026-postcatalysis-ppi","ref:yi2026"],"tags":["trigger-loop","pyrophosphate"],"difficulty":2,"url":"/machines/rna-polymerase#mechanism","cites":[{"source":"step:The trigger loop opens","machine":"rna-polymerase","label":"Step: The trigger loop opens","section":"mechanism"},{"source":"evidence:yi2026-ppi-bound-closed-loop","machine":"rna-polymerase","label":"Pyrophosphate in the product state (Yi G 2026)","section":"evidence","anchor":"ev-yi2026-ppi-bound-closed-loop"},{"source":"evidence:li2026-postcatalysis-ppi","machine":"rna-polymerase","label":"Pyrophosphate after the bond forms, peer-reviewed structure (Li Q 2026)","section":"evidence","anchor":"ev-li2026-postcatalysis-ppi"},{"source":"ref:yi2026","machine":"rna-polymerase","label":"Yi et al.","section":"sources","href":"https://doi.org/10.64898/2026.06.04.730248"}]},{"id":"rna-polymerase-brownian-ratchet","machine":"rna-polymerase","kind":"cloze","prompt":"In the Brownian ratchet model, RNA polymerase slides back and forth along the DNA by thermal motion, and {{an incoming NTP}} catches it in the forward position.","answer":"an incoming NTP","explanation":"Each cycle moves the DNA and RNA one base pair; the trigger loop swings between open, wedged and closed positions as part of this motion.","section":"mechanism","topic":"cycle","sources":["step:DNA and RNA move one base pair","mechanism:Translocation","ref:abbondanzieri2005"],"tags":["translocation","ratchet"],"difficulty":2,"url":"/machines/rna-polymerase#mechanism","cites":[{"source":"step:DNA and RNA move one base pair","machine":"rna-polymerase","label":"Step: DNA and RNA move one base pair","section":"mechanism"},{"source":"mechanism:Translocation","machine":"rna-polymerase","label":"Step: Translocation","section":"mechanism"},{"source":"ref:abbondanzieri2005","machine":"rna-polymerase","label":"Abbondanzieri et al.","section":"sources","href":"https://doi.org/10.1038/nature04268"}]},{"id":"rna-polymerase-proofreading-backtrack","machine":"rna-polymerase","kind":"qa","prompt":"How does RNA polymerase II remove a wrong base it has just added?","answer":"It backtracks one position, and the same active site cuts off the RNA end that holds the error.","explanation":"The wrong base first frays away from the template and the enzyme pauses. DNA polymerases, in contrast, use a separate nuclease site.","section":"mechanism","topic":"cycle","sources":["mechanism:Proofreading","stat:Proofreading","evolution:Proofreading in one active site","ref:sydow2009mc"],"tags":["proofreading","fidelity"],"difficulty":2,"url":"/machines/rna-polymerase#mechanism","cites":[{"source":"mechanism:Proofreading","machine":"rna-polymerase","label":"Step: Proofreading","section":"mechanism"},{"source":"stat:Proofreading","machine":"rna-polymerase","label":"Key number: Proofreading","section":"summary"},{"source":"evolution:Proofreading in one active site","machine":"rna-polymerase","label":"Proofreading in one active site","section":"summary"},{"source":"ref:sydow2009mc","machine":"rna-polymerase","label":"Sydow et al.","section":"sources","anchor":"ref-sydow2009mc","href":"https://doi.org/10.1016/j.molcel.2009.06.002"}]},{"id":"rna-polymerase-tfiis-rescue","machine":"rna-polymerase","kind":"cloze","prompt":"The factor {{TFIIS}} rescues backtracked RNA polymerase II by helping it cut its RNA.","answer":"TFIIS","explanation":"In a backtracked enzyme the RNA end runs into the pore and traps the trigger loop; TFIIS reaches into the active site, pushes the RNA out and helps cut it.","section":"mechanism","topic":"parts","sources":["step:The next base loads","evidence:backtracked-rna-site","ref:cheung2011"],"tags":["backtracking","proofreading"],"difficulty":2,"url":"/machines/rna-polymerase#mechanism","cites":[{"source":"step:The next base loads","machine":"rna-polymerase","label":"Step: The next base loads","section":"mechanism"},{"source":"evidence:backtracked-rna-site","machine":"rna-polymerase","label":"Backtracked RNA bound in arrested Pol II (Cheung AC 2011)","section":"evidence","anchor":"ev-backtracked-rna-site"},{"source":"ref:cheung2011","machine":"rna-polymerase","label":"Cheung and Cramer, Nature 2011","section":"sources","anchor":"ref-cheung2011","href":"https://doi.org/10.1038/nature09785"}]},{"id":"rna-polymerase-same-genome-different-cells","machine":"rna-polymerase","kind":"qa","prompt":"A skin cell and a nerve cell carry the same genome. What makes them differ, in terms of what RNA polymerase II copies?","answer":"Which genes (pages) they read.","explanation":"Every cell with a nucleus holds the full set of instructions; RNA polymerase II copies only the genes that cell needs.","section":"story","topic":"purpose","sources":["stop:body","analogy:A scribe copying one page from a huge library"],"tags":["purpose","genome"],"difficulty":1,"url":"/machines/rna-polymerase#story","cites":[{"source":"stop:body","machine":"rna-polymerase","label":"Big picture: You, a library","section":"story"},{"source":"analogy:A scribe copying one page from a huge library","machine":"rna-polymerase","label":"Analogy: A scribe copying one page from a huge library","section":"story"}]},{"id":"rna-polymerase-dystrophin-time","machine":"rna-polymerase","kind":"cloze","prompt":"One RNA polymerase II takes about {{16 hours}} to copy the human dystrophin gene once.","answer":"16 hours","explanation":"Dystrophin is at least 2,300 kb long; muscle cell cultures copied about 1,770 kb in about 12 h (about 2.4 kb per minute), and the authors extrapolated.","section":"story","topic":"numbers","sources":["fact:V9","stop:gene","ref:tennyson1995"],"tags":["speed","genes"],"difficulty":2,"url":"/machines/rna-polymerase#story","cites":[{"source":"fact:V9","machine":"rna-polymerase","label":"Time to copy the dystrophin gene once: 16 h","section":"story"},{"source":"stop:gene","machine":"rna-polymerase","label":"Big picture: One long gene","section":"story"},{"source":"ref:tennyson1995","machine":"rna-polymerase","label":"Tennyson 1995","section":"sources","href":"https://doi.org/10.1038/ng0295-184"}]},{"id":"rna-polymerase-copies-per-hela","machine":"rna-polymerase","kind":"qa","prompt":"About how many RNA polymerase II molecules does one HeLa cell hold?","answer":"About 320,000.","explanation":"About 65,000 (roughly 20%) copy a gene at one time, but the source cites that share from earlier work rather than measuring it.","section":"story","topic":"numbers","sources":["fact:V12","ref:kimura1999"],"tags":["copy-number"],"difficulty":2,"url":"/machines/rna-polymerase#story","cites":[{"source":"fact:V12","machine":"rna-polymerase","label":"RNA polymerase II molecules in one HeLa cell: 320,000","section":"story"},{"source":"ref:kimura1999","machine":"rna-polymerase","label":"Kimura 1999","section":"sources","href":"https://doi.org/10.1128/mcb.19.8.5383"}]},{"id":"rna-polymerase-ribosome-link","machine":"rna-polymerase","kind":"cloze","prompt":"In a fast-growing yeast cell, {{half}} of all RNA polymerase II transcription serves the genes for ribosomal proteins.","answer":"half","explanation":"Ribosomes read the messenger RNA that RNA polymerase II writes.","section":"story","topic":"purpose","sources":["link:ribosome","ref:warner1999"],"tags":["ribosome","links"],"difficulty":2,"url":"/machines/rna-polymerase#story","cites":[{"source":"link:ribosome","machine":"rna-polymerase","label":"Link to Ribosome","section":"story"},{"source":"ref:warner1999","machine":"rna-polymerase","label":"Warner 1999","section":"sources","href":"https://doi.org/10.1016/s0968-0004(99)01460-7"}]},{"id":"rna-polymerase-train-analogy-breaks","machine":"rna-polymerase","kind":"qa","prompt":"Where does the \"train on a track\" analogy for RNA polymerase II break down?","answer":"Pol II pauses, backs up to fix errors, and speeds up and slows down along a gene.","explanation":"The analogy gets one thing right: Pol II runs along the gene in one direction.","section":"story","topic":"cycle","sources":["analogy:A train on a track","stat:Elongation rate, genome-wide","ref:jonkers2014"],"tags":["analogy","speed"],"difficulty":2,"url":"/machines/rna-polymerase#story","cites":[{"source":"analogy:A train on a track","machine":"rna-polymerase","label":"Analogy: A train on a track","section":"story"},{"source":"stat:Elongation rate, genome-wide","machine":"rna-polymerase","label":"Key number: Elongation rate, genome-wide","section":"summary"},{"source":"ref:jonkers2014","machine":"rna-polymerase","label":"Jonkers et al.","section":"sources","anchor":"ref-jonkers2014","href":"https://doi.org/10.7554/elife.02407"}]},{"id":"rna-polymerase-rate-long-genes","machine":"rna-polymerase","kind":"cloze","prompt":"Over long human genes, RNA polymerase II copies about {{3.8 kb per minute}}, about 63 nucleotides per second.","answer":"3.8 kb per minute","explanation":"Measured in human Tet-21 cells: 3.79 ± 0.26 kb per minute over 15 gene regions. That is about 16 ms per nucleotide.","section":"evidence","topic":"numbers","sources":["evidence:elongation-rate-long-human-genes","stat:Elongation rate in human cells","step:The addition site is open","ref:singh2009"],"tags":["speed"],"difficulty":1,"url":"/machines/rna-polymerase#evidence","cites":[{"source":"evidence:elongation-rate-long-human-genes","machine":"rna-polymerase","label":"Pol II elongation rate over long endogenous human genes (Singh J 2009)","section":"evidence","anchor":"ev-elongation-rate-long-human-genes"},{"source":"stat:Elongation rate in human cells","machine":"rna-polymerase","label":"Key number: Elongation rate in human cells","section":"summary"},{"source":"step:The addition site is open","machine":"rna-polymerase","label":"Step: The addition site is open","section":"mechanism"},{"source":"ref:singh2009","machine":"rna-polymerase","label":"Singh and Padgett, Nature Structural and Molecular Biology 2009","section":"sources","anchor":"ref-singh2009","href":"https://doi.org/10.1038/nsmb.1666"}]},{"id":"rna-polymerase-rate-method","machine":"rna-polymerase","kind":"qa","prompt":"How did Singh and Padgett measure the speed of RNA polymerase II over long human genes?","answer":"They blocked new transcription with DRB, washed it out, and timed when new pre-mRNA reached exon-intron junctions far apart in the same gene.","explanation":"The delay between two junctions, read by RT-PCR, gives the speed; it was about the same over long introns and exon-rich stretches.","section":"evidence","topic":"numbers","sources":["evidence:elongation-rate-long-human-genes","ref:singh2009"],"tags":["speed","method"],"difficulty":3,"url":"/machines/rna-polymerase#evidence","cites":[{"source":"evidence:elongation-rate-long-human-genes","machine":"rna-polymerase","label":"Pol II elongation rate over long endogenous human genes (Singh J 2009)","section":"evidence","anchor":"ev-elongation-rate-long-human-genes"},{"source":"ref:singh2009","machine":"rna-polymerase","label":"Singh and Padgett, Nature Structural and Molecular Biology 2009","section":"sources","anchor":"ref-singh2009","href":"https://doi.org/10.1038/nsmb.1666"}]},{"id":"rna-polymerase-error-rate","machine":"rna-polymerase","kind":"qa","prompt":"About what transcription error rate per base was measured across all mRNA of the worm Caenorhabditis elegans?","answer":"About 4 x 10^-6 per base.","explanation":"That is about four mistakes per million letters (4.1 × 10^-6 pooled over three strains).","section":"evidence","topic":"numbers","sources":["evidence:transcription-error-rate","stat:Transcription error rate","fact:V11","ref:gout2013"],"tags":["fidelity"],"difficulty":2,"url":"/machines/rna-polymerase#evidence","cites":[{"source":"evidence:transcription-error-rate","machine":"rna-polymerase","label":"Base-substitution error rate in mRNA in vivo (Gout JF 2013)","section":"evidence","anchor":"ev-transcription-error-rate"},{"source":"stat:Transcription error rate","machine":"rna-polymerase","label":"Key number: Transcription error rate","section":"summary"},{"source":"fact:V11","machine":"rna-polymerase","label":"Transcription errors: 4 × 10^-6 per letter","section":"story"},{"source":"ref:gout2013","machine":"rna-polymerase","label":"Gout et al.","section":"sources","anchor":"ref-gout2013","href":"https://doi.org/10.1073/pnas.1309843110"}]},{"id":"rna-polymerase-error-rate-method","machine":"rna-polymerase","kind":"qa","prompt":"How did Gout and colleagues tell real transcription errors apart from errors made while copying and sequencing the RNA?","answer":"They tagged each RNA fragment, copied it three times, and counted a change only if every copy carried it.","explanation":"Requiring the change in every copy removes errors made during copying and sequencing.","section":"evidence","topic":"numbers","sources":["evidence:transcription-error-rate","ref:gout2013"],"tags":["fidelity","method"],"difficulty":3,"url":"/machines/rna-polymerase#evidence","cites":[{"source":"evidence:transcription-error-rate","machine":"rna-polymerase","label":"Base-substitution error rate in mRNA in vivo (Gout JF 2013)","section":"evidence","anchor":"ev-transcription-error-rate"},{"source":"ref:gout2013","machine":"rna-polymerase","label":"Gout et al.","section":"sources","anchor":"ref-gout2013","href":"https://doi.org/10.1073/pnas.1309843110"}]},{"id":"rna-polymerase-mismatch-slows-next","machine":"rna-polymerase","kind":"qa","prompt":"According to Thomas and colleagues, what delay, more than the slow addition of the wrong base itself, lets RNA polymerase II tell right from wrong?","answer":"The next nucleotide is added at least 15- to 20-fold more slowly after a mismatched RNA end.","explanation":"The pause gives the enzyme time to back up and cut out the error. Measured with Pol II complexes from human nuclear extract.","section":"evidence","topic":"cycle","sources":["evidence:mismatch-slows-next-addition","evidence:misincorporation-slower-than-correct","ref:thomas1998"],"tags":["fidelity","proofreading"],"difficulty":3,"url":"/machines/rna-polymerase#evidence","cites":[{"source":"evidence:mismatch-slows-next-addition","machine":"rna-polymerase","label":"Extension rate after a mismatched RNA 3' end (Thomas MJ 1998)","section":"evidence","anchor":"ev-mismatch-slows-next-addition"},{"source":"evidence:misincorporation-slower-than-correct","machine":"rna-polymerase","label":"Rate of correct versus incorrect nucleotide incorporation (Thomas MJ 1998)","section":"evidence","anchor":"ev-misincorporation-slower-than-correct"},{"source":"ref:thomas1998","machine":"rna-polymerase","label":"Thomas et al.","section":"sources","anchor":"ref-thomas1998","href":"https://doi.org/10.1016/S0092-8674(00)81191-5"}]},{"id":"rna-polymerase-deoxy-ntp-chemistry","machine":"rna-polymerase","kind":"qa","prompt":"How did Wang and colleagues show that RNA polymerase II rejects 2′-deoxy NTPs mainly at the chemical step, not at binding?","answer":"Deoxy NTPs were added at least 400-fold more slowly, while their KM values were much closer to normal.","explanation":"A deoxy NTP still binds the addition site, so binding alone cannot tell it from an RNA building block; trigger-loop closure and the chemistry do.","section":"evidence","topic":"cycle","sources":["evidence:deoxy-ntp-discrimination","step:The NTP pairs with the template","ref:wang2006"],"tags":["fidelity","ntp"],"difficulty":3,"url":"/machines/rna-polymerase#evidence","cites":[{"source":"evidence:deoxy-ntp-discrimination","machine":"rna-polymerase","label":"Rate of 2'-deoxy NTP addition (Wang D 2006)","section":"evidence","anchor":"ev-deoxy-ntp-discrimination"},{"source":"step:The NTP pairs with the template","machine":"rna-polymerase","label":"Step: The NTP pairs with the template","section":"mechanism"},{"source":"ref:wang2006","machine":"rna-polymerase","label":"Wang et al.","section":"sources","anchor":"ref-wang2006","href":"https://doi.org/10.1016/j.cell.2006.11.023"}]},{"id":"rna-polymerase-ppi-power-stroke-debate","machine":"rna-polymerase","kind":"qa","prompt":"Which model of RNA polymerase movement did optical-trap force-velocity data favour over a power stroke tied to pyrophosphate release?","answer":"A Brownian ratchet with a second NTP site.","explanation":"The power stroke fitted poorly (reduced χ² 6.03), measured on E. coli RNA polymerase. The machine notes still say pyrophosphate release drives the chain forward.","section":"evidence","topic":"debate","sources":["evidence:brownian-ratchet-force-velocity","step:Pyrophosphate leaves","machine:energy","ref:abbondanzieri2005"],"tags":["ratchet","pyrophosphate"],"difficulty":3,"url":"/machines/rna-polymerase#evidence","cites":[{"source":"evidence:brownian-ratchet-force-velocity","machine":"rna-polymerase","label":"Mechanism that fits the force-velocity data (Abbondanzieri EA 2005)","section":"evidence","anchor":"ev-brownian-ratchet-force-velocity"},{"source":"step:Pyrophosphate leaves","machine":"rna-polymerase","label":"Step: Pyrophosphate leaves","section":"mechanism"},{"source":"machine:energy","machine":"rna-polymerase","label":"Summary","section":"summary"},{"source":"ref:abbondanzieri2005","machine":"rna-polymerase","label":"Abbondanzieri et al.","section":"sources","href":"https://doi.org/10.1038/nature04268"}]},{"id":"groel-what-it-does","machine":"groel","kind":"qa","prompt":"What does GroEL do for a protein that has not folded?","answer":"It closes the protein in a chamber where it folds alone, safe from aggregation.","explanation":"Hydrophobic surfaces in one ring catch the protein; ATP and the lid GroES then close the chamber around it.","section":"summary","topic":"purpose","sources":["machine:summary","mechanism:Folding in isolation","ref:weissman1996"],"tags":["folding","aggregation"],"difficulty":1,"url":"/machines/groel#summary","cites":[{"source":"machine:summary","machine":"groel","label":"Summary","section":"summary"},{"source":"mechanism:Folding in isolation","machine":"groel","label":"Step: Folding in isolation","section":"mechanism"},{"source":"ref:weissman1996","machine":"groel","label":"Weissman et al.","section":"sources","anchor":"ref-weissman1996","href":"https://doi.org/10.1016/s0092-8674(00)81293-3"}]},{"id":"groel-equatorial-domain-role","machine":"groel","kind":"qa","prompt":"Which GroEL domain binds ATP and holds the two rings together?","answer":"The equatorial domain.","explanation":"Each GroEL subunit has three domains: equatorial (ATP, ring contacts), intermediate (the hinge) and apical (binds substrate and GroES).","section":"summary","topic":"parts","sources":["component:GroEL equatorial domain","evidence:groel-fourteen-subunits","ref:braig1994"],"tags":["domains","atp"],"difficulty":1,"url":"/machines/groel#summary","cites":[{"source":"component:GroEL equatorial domain","machine":"groel","label":"Part: GroEL equatorial domain","section":"summary"},{"source":"evidence:groel-fourteen-subunits","machine":"groel","label":"Subunit architecture of GroEL (Braig K 1994)","section":"evidence","anchor":"ev-groel-fourteen-subunits"},{"source":"ref:braig1994","machine":"groel","label":"Braig et al.","section":"sources","anchor":"ref-braig1994","href":"https://doi.org/10.1038/371578a0"}]},{"id":"groel-apical-helices-h-i","machine":"groel","kind":"qa","prompt":"Through which helices does the GroEL apical domain bind both the unfolded substrate and GroES?","answer":"Helices H and I.","explanation":"The same sites hold the client first and GroES later, so GroES binding takes the sites away from the client.","section":"summary","topic":"parts","sources":["component:GroEL apical domain","step:GroES closes the new chamber","ref:clare2012"],"tags":["domains","binding"],"difficulty":2,"url":"/machines/groel#summary","cites":[{"source":"component:GroEL apical domain","machine":"groel","label":"Part: GroEL apical domain","section":"summary"},{"source":"step:GroES closes the new chamber","machine":"groel","label":"Step: GroES closes the new chamber","section":"mechanism"},{"source":"ref:clare2012","machine":"groel","label":"Clare et al.","section":"sources","anchor":"ref-clare2012","href":"https://doi.org/10.1016/j.cell.2012.02.047"}]},{"id":"groel-thermosome-built-in-lid","machine":"groel","kind":"qa","prompt":"Why does the archaeal thermosome, a group II chaperonin, need no GroES lid?","answer":"Its own apical domains form a built-in lid.","explanation":"Group I chaperonins such as GroEL, in bacteria and organelles, use a separate GroES lid. The thermosome also has eight subunits per ring, not seven.","section":"summary","topic":"parts","sources":["species:Thermosome","evolution:Two chaperonin groups","ref:ditzel1998"],"tags":["evolution","lid"],"difficulty":2,"url":"/machines/groel#summary","cites":[{"source":"species:Thermosome","machine":"groel","label":"Thermosome","section":"summary"},{"source":"evolution:Two chaperonin groups","machine":"groel","label":"Two chaperonin groups","section":"summary"},{"source":"ref:ditzel1998","machine":"groel","label":"Ditzel et al.","section":"sources","anchor":"ref-ditzel1998","href":"https://doi.org/10.1016/s0092-8674(00)81152-6"}]},{"id":"groel-seven-per-ring","machine":"groel","kind":"cloze","prompt":"GroEL is a cylinder of two stacked rings, each made of {{seven}} subunits.","answer":"seven","explanation":"That gives 14 GroEL subunits, capped by one GroES ring of 7 subunits (X-ray structure, Braig et al. 1994).","section":"summary","topic":"numbers","sources":["stat:Subunits","evidence:groel-fourteen-subunits","ref:braig1994"],"tags":["structure"],"difficulty":1,"url":"/machines/groel#summary","cites":[{"source":"stat:Subunits","machine":"groel","label":"Key number: Subunits","section":"summary"},{"source":"evidence:groel-fourteen-subunits","machine":"groel","label":"Subunit architecture of GroEL (Braig K 1994)","section":"evidence","anchor":"ev-groel-fourteen-subunits"},{"source":"ref:braig1994","machine":"groel","label":"Braig et al.","section":"sources","anchor":"ref-braig1994","href":"https://doi.org/10.1038/371578a0"}]},{"id":"groel-size-limit","machine":"groel","kind":"cloze","prompt":"The closed GroEL–GroES chamber can hold unfolded proteins up to about {{60 kDa}}.","answer":"60 kDa","explanation":"Denatured proteins smaller than the 57 kDa GroEL subunit stay inside; an 82 kDa protein binds GroEL but never enters.","section":"summary","topic":"numbers","sources":["stat:Substrate size limit","ref:sakikawa1999"],"tags":["chamber","size"],"difficulty":2,"url":"/machines/groel#summary","cites":[{"source":"stat:Substrate size limit","machine":"groel","label":"Key number: Substrate size limit","section":"summary"},{"source":"ref:sakikawa1999","machine":"groel","label":"Sakikawa et al.","section":"sources","anchor":"ref-sakikawa1999","href":"https://doi.org/10.1074/jbc.274.30.21251"}]},{"id":"groel-atp-per-cycle","machine":"groel","kind":"qa","prompt":"How many ATP does one GroEL ring bind and hydrolyse in each folding cycle?","answer":"7","explanation":"One ATP per subunit of the ring. Each turn of the alternating-ring cycle uses one ringful of ATP (Rye et al. 1999).","section":"summary","topic":"numbers","sources":["stat:ATP per folding cycle","machine:energy","ref:rye1999"],"tags":["atp"],"difficulty":1,"url":"/machines/groel#summary","cites":[{"source":"stat:ATP per folding cycle","machine":"groel","label":"Key number: ATP per folding cycle","section":"summary"},{"source":"machine:energy","machine":"groel","label":"Summary","section":"summary"},{"source":"ref:rye1999","machine":"groel","label":"Rye et al.","section":"sources","anchor":"ref-rye1999","href":"https://doi.org/10.1016/s0092-8674(00)80742-4"}]},{"id":"groel-evolved-specialist-cost","machine":"groel","kind":"qa","prompt":"What did GroEL/GroES variants evolved to fold green fluorescent protein lose in return?","answer":"General folding ability.","explanation":"The best variant made cells 8-fold brighter with GFP but cut lambda phage plaque formation about 1000-fold. A specialist cage trades away the generalist one.","section":"summary","topic":"debate","sources":["frontier:Chaperonins evolved for one substrate","evidence:evolved-groe-folds-gfp","ref:wang2002"],"tags":["evolution","engineering"],"difficulty":2,"url":"/machines/groel#summary","cites":[{"source":"frontier:Chaperonins evolved for one substrate","machine":"groel","label":"Open question: Chaperonins evolved for one substrate","section":"summary"},{"source":"evidence:evolved-groe-folds-gfp","machine":"groel","label":"Gain and cost of a GFP-optimized GroEL/GroES (Wang JD 2002)","section":"evidence","anchor":"ev-evolved-groe-folds-gfp"},{"source":"ref:wang2002","machine":"groel","label":"Wang et al.","section":"sources","anchor":"ref-wang2002","href":"https://doi.org/10.1016/s0092-8674(02)01198-4"}]},{"id":"groel-capture-hydrophobic","machine":"groel","kind":"qa","prompt":"What does an unfolded protein expose that lets the open GroEL ring catch it?","answer":"Water-repelling (hydrophobic) stretches.","explanation":"These stretches bind a water-repelling collar formed by helices H and I of all seven subunits, with several contacts at once.","section":"mechanism","topic":"cycle","sources":["step:An unfolded protein is caught","mechanism:Capture","ref:braig1994"],"tags":["capture","hydrophobic"],"difficulty":2,"url":"/machines/groel#mechanism","cites":[{"source":"step:An unfolded protein is caught","machine":"groel","label":"Step: An unfolded protein is caught","section":"mechanism"},{"source":"mechanism:Capture","machine":"groel","label":"Step: Capture","section":"mechanism"},{"source":"ref:braig1994","machine":"groel","label":"Braig et al.","section":"sources","anchor":"ref-braig1994","href":"https://doi.org/10.1038/371578a0"}]},{"id":"groel-ring-anticooperativity","machine":"groel","kind":"cloze","prompt":"In GroEL, ATP binding is cooperative within a ring but {{strongly anti-cooperative}} between the two rings.","answer":"strongly anti-cooperative","explanation":"Yifrach and Horovitz fitted an inter-ring Hill coefficient of 0.003. So a ring binds its seven ATP together, and the two rings do not do so at the same time.","section":"mechanism","topic":"cycle","sources":["step:ADP leaves and seven ATP bind","evidence:nested-allostery"],"tags":["atp","allostery"],"difficulty":3,"url":"/machines/groel#mechanism","cites":[{"source":"step:ADP leaves and seven ATP bind","machine":"groel","label":"Step: ADP leaves and seven ATP bind","section":"mechanism"},{"source":"evidence:nested-allostery","machine":"groel","label":"Cooperativity of ATP hydrolysis within and between rings (Yifrach O 1995)","section":"evidence","anchor":"ev-nested-allostery"}]},{"id":"groel-apical-rise-stretches","machine":"groel","kind":"cloze","prompt":"As the GroEL apical domains rise after ATP binds, they stretch the bound protein, which can {{pull misfolded parts apart}}.","answer":"pull misfolded parts apart","explanation":"The apical domains rise and move outwards, so the protein is pulled between its contact points.","section":"mechanism","topic":"cycle","sources":["mechanism:The apical domains rise","step:The apical domains tilt and rise","ref:clare2012"],"tags":["apical","unfolding"],"difficulty":2,"url":"/machines/groel#mechanism","cites":[{"source":"mechanism:The apical domains rise","machine":"groel","label":"Step: The apical domains rise","section":"mechanism"},{"source":"step:The apical domains tilt and rise","machine":"groel","label":"Step: The apical domains tilt and rise","section":"mechanism"},{"source":"ref:clare2012","machine":"groel","label":"Clare et al.","section":"sources","anchor":"ref-clare2012","href":"https://doi.org/10.1016/j.cell.2012.02.047"}]},{"id":"groel-what-opens-old-chamber","machine":"groel","kind":"qa","prompt":"In GroEL, what makes the closed chamber on one ring open and release GroES?","answer":"ATP (with a new client) binding to the opposite ring.","explanation":"The signal crosses the ring contact through the equatorial domains. The old ring lets go of GroES and its protein, folded or not, so the two rings alternate.","section":"mechanism","topic":"cycle","sources":["mechanism:The other ring fires","step:The old chamber opens","ref:rye1999"],"tags":["alternation","release"],"difficulty":2,"url":"/machines/groel#mechanism","cites":[{"source":"mechanism:The other ring fires","machine":"groel","label":"Step: The other ring fires","section":"mechanism"},{"source":"step:The old chamber opens","machine":"groel","label":"Step: The old chamber opens","section":"mechanism"},{"source":"ref:rye1999","machine":"groel","label":"Rye et al.","section":"sources","anchor":"ref-rye1999","href":"https://doi.org/10.1016/s0092-8674(00)80742-4"}]},{"id":"groel-power-stroke-twist","machine":"groel","kind":"qa","prompt":"When GroES docks on GroEL, what does the final twist of about 100° of the apical domains do to the bound client?","answer":"It peels the water-repelling sites off the client and drops it into the chamber.","explanation":"Clare and colleagues call this twist the power stroke of GroEL. It buries the sites against GroES and doubles the chamber volume.","section":"mechanism","topic":"cycle","sources":["step:GroES closes the new chamber","evidence:apical-domain-twist","mechanism:GroES docks and the cage closes","ref:clare2012","ref:xu1997"],"tags":["power-stroke","apical"],"difficulty":3,"url":"/machines/groel#mechanism","cites":[{"source":"step:GroES closes the new chamber","machine":"groel","label":"Step: GroES closes the new chamber","section":"mechanism"},{"source":"evidence:apical-domain-twist","machine":"groel","label":"Final apical-domain rotation that docks GroES (Clare DK 2012)","section":"evidence","anchor":"ev-apical-domain-twist"},{"source":"mechanism:GroES docks and the cage closes","machine":"groel","label":"Step: GroES docks and the cage closes","section":"mechanism"},{"source":"ref:clare2012","machine":"groel","label":"Clare et al.","section":"sources","anchor":"ref-clare2012","href":"https://doi.org/10.1016/j.cell.2012.02.047"},{"source":"ref:xu1997","machine":"groel","label":"Xu et al.","section":"sources","anchor":"ref-xu1997","href":"https://doi.org/10.1038/41944"}]},{"id":"groel-why-no-aggregation-inside","machine":"groel","kind":"qa","prompt":"Why can a protein folding inside the closed GroEL chamber not aggregate?","answer":"The chamber holds only that one protein, so it meets no other chains.","explanation":"The chamber lining is also water-loving, so the folding protein finds nothing sticky.","section":"mechanism","topic":"cycle","sources":["mechanism:Folding in isolation","step:One ring is a closed chamber","ref:weissman1996"],"tags":["chamber","aggregation"],"difficulty":1,"url":"/machines/groel#mechanism","cites":[{"source":"mechanism:Folding in isolation","machine":"groel","label":"Step: Folding in isolation","section":"mechanism"},{"source":"step:One ring is a closed chamber","machine":"groel","label":"Step: One ring is a closed chamber","section":"mechanism"},{"source":"ref:weissman1996","machine":"groel","label":"Weissman et al.","section":"sources","anchor":"ref-weissman1996","href":"https://doi.org/10.1016/s0092-8674(00)81293-3"}]},{"id":"groel-hydrolysis-timer","machine":"groel","kind":"cloze","prompt":"In GroEL, {{ATP hydrolysis}} in the closed ring sets how long the chamber stays shut.","answer":"ATP hydrolysis","explanation":"The ADP state holds GroES less tightly (Rye et al. 1997), and only after hydrolysis can the opposite ring take up a client and GroES (Rye et al. 1999).","section":"mechanism","topic":"cycle","sources":["step:ATP is split: the timer","mechanism:Hydrolysis primes release","ref:rye1997","ref:rye1999"],"tags":["atp","timer"],"difficulty":2,"url":"/machines/groel#mechanism","cites":[{"source":"step:ATP is split: the timer","machine":"groel","label":"Step: ATP is split: the timer","section":"mechanism"},{"source":"mechanism:Hydrolysis primes release","machine":"groel","label":"Step: Hydrolysis primes release","section":"mechanism"},{"source":"ref:rye1997","machine":"groel","label":"Rye et al.","section":"sources","anchor":"ref-rye1997","href":"https://doi.org/10.1038/42047"},{"source":"ref:rye1999","machine":"groel","label":"Rye et al.","section":"sources","anchor":"ref-rye1999","href":"https://doi.org/10.1016/s0092-8674(00)80742-4"}]},{"id":"groel-stuck-chains-clump","machine":"groel","kind":"qa","prompt":"In E. coli, why is a new protein chain that gets stuck half-folded a problem for the cell?","answer":"Its sticky patches can stick to other stuck chains and form a useless clump.","explanation":"Such clumps are useless and can be toxic. GroEL catches stuck chains so they get another chance to fold.","section":"story","topic":"purpose","sources":["stop:chain","story:summary"],"tags":["aggregation","cell"],"difficulty":1,"url":"/machines/groel#story","cites":[{"source":"stop:chain","machine":"groel","label":"Big picture: A new chain","section":"story"},{"source":"story:summary","machine":"groel","label":"Big picture","section":"story"}]},{"id":"groel-share-of-proteins","machine":"groel","kind":"cloze","prompt":"In E. coli during normal growth, about {{10–15%}} of cytoplasmic protein passes through GroEL.","answer":"10–15%","explanation":"Under heat stress the share rises to 30% or more. Most proteins leave GroEL within 10–30 s.","section":"story","topic":"numbers","sources":["fact:G4","ref:ewalt1997"],"tags":["cell","flux"],"difficulty":2,"url":"/machines/groel#story","cites":[{"source":"fact:G4","machine":"groel","label":"Share of cytoplasmic protein that passes through GroEL: 10–15 %","section":"story"},{"source":"ref:ewalt1997","machine":"groel","label":"Ewalt 1997","section":"sources","href":"https://doi.org/10.1016/s0092-8674(00)80509-7"}]},{"id":"groel-essential-for-growth","machine":"groel","kind":"qa","prompt":"How well does E. coli grow without GroEL and GroES?","answer":"Not at all: it cannot grow at any temperature tested.","explanation":"About 85 E. coli proteins need GroEL to fold, and 13 of those are essential to the cell.","section":"story","topic":"purpose","sources":["fact:G8","fact:G7","ref:fayet1989","ref:kerner2005"],"tags":["essential","cell"],"difficulty":1,"url":"/machines/groel#story","cites":[{"source":"fact:G8","machine":"groel","label":"Temperatures at which E. coli needs GroEL and GroES to grow: 17–42 °C","section":"story"},{"source":"fact:G7","machine":"groel","label":"E. coli proteins that bind GroEL: 250 of about 2,400 cytosolic proteins","section":"story"},{"source":"ref:fayet1989","machine":"groel","label":"Fayet 1989","section":"sources","href":"https://doi.org/10.1128/jb.171.3.1379-1385.1989"},{"source":"ref:kerner2005","machine":"groel","label":"Kerner et al.","section":"sources","anchor":"ref-kerner2005","href":"https://doi.org/10.1016/j.cell.2005.05.028"}]},{"id":"groel-human-hsp60","machine":"groel","kind":"qa","prompt":"What is the GroEL relative in human mitochondria called?","answer":"Hsp60","explanation":"Mitochondria descend from bacteria and kept this folding chamber. Human Hsp60 with its lid, Hsp10, lets E. coli grow without its own GroEL and GroES.","section":"story","topic":"parts","sources":["fact:G9","ref:cheng1989","ref:hansen2002"],"tags":["mitochondria","evolution"],"difficulty":1,"url":"/machines/groel#story","cites":[{"source":"fact:G9","machine":"groel","label":"Human relative of GroEL: Hsp60 in mitochondria","section":"story"},{"source":"ref:cheng1989","machine":"groel","label":"Cheng 1989","section":"sources","href":"https://doi.org/10.1038/337620a0"},{"source":"ref:hansen2002","machine":"groel","label":"Hansen 2002","section":"sources","href":"https://doi.org/10.1086/339935"}]},{"id":"groel-quiet-room-open-question","machine":"groel","kind":"qa","prompt":"The GroEL chamber is often pictured as \"a quiet room for one\". What does that picture leave open?","answer":"Whether the chamber actively helps the fold, beyond keeping other chains out.","explanation":"This is debated (Hayer-Hartl et al. 2016; Horwich et al. 2009). The room also changes: its walls are sticky while catching the chain and water-loving once the lid closes.","section":"story","topic":"debate","sources":["analogy:A quiet room for one","ref:hayerhartl2016","ref:horwich2009"],"tags":["analogy","debate"],"difficulty":3,"url":"/machines/groel#story","cites":[{"source":"analogy:A quiet room for one","machine":"groel","label":"Analogy: A quiet room for one","section":"story"},{"source":"ref:hayerhartl2016","machine":"groel","label":"Hayer-Hartl et al.","section":"sources","anchor":"ref-hayerhartl2016","href":"https://doi.org/10.1016/j.tibs.2015.07.009"},{"source":"ref:horwich2009","machine":"groel","label":"Horwich et al.","section":"sources","anchor":"ref-horwich2009","href":"https://doi.org/10.1017/s0033583509004764"}]},{"id":"groel-second-chance-by-chance","machine":"groel","kind":"qa","prompt":"How does a chain that GroEL releases still unfolded get another round of folding?","answer":"It simply binds GroEL again by chance; GroEL does not check the result.","explanation":"Of about 300 new proteins that bind GroEL strongly, about one third are unstable and return to it again and again.","section":"story","topic":"cycle","sources":["analogy:A second chance","fact:G6","ref:houry1999"],"tags":["analogy","rebinding"],"difficulty":2,"url":"/machines/groel#story","cites":[{"source":"analogy:A second chance","machine":"groel","label":"Analogy: A second chance","section":"story"},{"source":"fact:G6","machine":"groel","label":"New proteins that bind GroEL strongly: 300 proteins","section":"story"},{"source":"ref:houry1999","machine":"groel","label":"Houry 1999","section":"sources","href":"https://doi.org/10.1038/45977"}]},{"id":"groel-size-limit-method","machine":"groel","kind":"qa","prompt":"How did Sakikawa and colleagues (1999) find which unfolded E. coli proteins fit inside the closed GroEL–GroES cage?","answer":"They closed GroES over the captured proteins and digested everything outside with protease; only proteins inside survived.","explanation":"Only proteins smaller than one 57 kDa GroEL subunit survived. An 82 kDa GFP trimer bound GroEL but was always digested, so it never entered.","section":"evidence","topic":"numbers","sources":["evidence:cis-cavity-size-limit","evidence:gfp-trimer-excluded","ref:sakikawa1999"],"tags":["method","size"],"difficulty":2,"url":"/machines/groel#evidence","cites":[{"source":"evidence:cis-cavity-size-limit","machine":"groel","label":"Largest protein held in the closed cis cavity (Sakikawa C 1999)","section":"evidence","anchor":"ev-cis-cavity-size-limit"},{"source":"evidence:gfp-trimer-excluded","machine":"groel","label":"Fate of an 82-kDa protein at GroEL-GroES (Sakikawa C 1999)","section":"evidence","anchor":"ev-gfp-trimer-excluded"},{"source":"ref:sakikawa1999","machine":"groel","label":"Sakikawa et al.","section":"sources","anchor":"ref-sakikawa1999","href":"https://doi.org/10.1074/jbc.274.30.21251"}]},{"id":"groel-hydrolysis-rates-differ","machine":"groel","kind":"qa","prompt":"ATP hydrolysis in the closed GroEL ring was measured at 0.12 s^-1 per subunit (Burston et al. 1995) and as a burst at 0.518 s^-1 (Ye and Lorimer 2013). Why do the two rates differ?","answer":"The studies used different methods and salt conditions.","explanation":"Burston and colleagues used transient kinetics after GroES binding; Ye and Lorimer followed phosphate release by stopped-flow at 0.2 M K+.","section":"evidence","topic":"debate","sources":["evidence:atp-hydrolysis-burst","evidence:atp-hydrolysis-in-cis-complex","stat:ATP hydrolysis rate","ref:ye2013","ref:burston1995"],"tags":["atp","kinetics"],"difficulty":3,"url":"/machines/groel#evidence","cites":[{"source":"evidence:atp-hydrolysis-burst","machine":"groel","label":"Pre-steady-state ATP hydrolysis and ADP release rates (Ye X 2013)","section":"evidence","anchor":"ev-atp-hydrolysis-burst"},{"source":"evidence:atp-hydrolysis-in-cis-complex","machine":"groel","label":"Rate of ATP hydrolysis in the GroEL-ATP7-GroES complex (Burston SG 1995)","section":"evidence","anchor":"ev-atp-hydrolysis-in-cis-complex"},{"source":"stat:ATP hydrolysis rate","machine":"groel","label":"Key number: ATP hydrolysis rate","section":"summary"},{"source":"ref:ye2013","machine":"groel","label":"Ye and Lorimer, PNAS 2013","section":"sources","href":"https://doi.org/10.1073/pnas.1317702110"},{"source":"ref:burston1995","machine":"groel","label":"Burston et al.","section":"sources","anchor":"ref-burston1995","href":"https://doi.org/10.1006/jmbi.1995.0285"}]},{"id":"groel-groes-stay-no-substrate","machine":"groel","kind":"qa","prompt":"Without substrate protein, about how long does the GroES lid stay on E. coli GroEL?","answer":"About 24 s.","explanation":"In trapping experiments GroES left at 0.042 s^-1 per subunit, the same as the steady-state ATPase rate; 24 s is 1 divided by that rate. Without substrate, GroES release is the slowest step.","section":"evidence","topic":"numbers","sources":["evidence:groes-release-steady-state","stat:GroES release rate","ref:burston1995"],"tags":["groes","kinetics"],"difficulty":2,"url":"/machines/groel#evidence","cites":[{"source":"evidence:groes-release-steady-state","machine":"groel","label":"GroES dissociation rate during steady-state cycling (Burston SG 1995)","section":"evidence","anchor":"ev-groes-release-steady-state"},{"source":"stat:GroES release rate","machine":"groel","label":"Key number: GroES release rate","section":"summary"},{"source":"ref:burston1995","machine":"groel","label":"Burston et al.","section":"sources","anchor":"ref-burston1995","href":"https://doi.org/10.1006/jmbi.1995.0285"}]},{"id":"groel-substrate-speeds-release","machine":"groel","kind":"cloze","prompt":"In stopped-flow FRET experiments, unfolded substrate protein sped up GroES release from GroEL {{20- to 50-fold}}.","answer":"20- to 50-fold","explanation":"The fast route grew with denatured Rubisco. Unfolded protein binds the open ring first and speeds release of the old cage, so GroEL spends its time folding rather than waiting.","section":"evidence","topic":"numbers","sources":["evidence:substrate-accelerates-groes-release","ref:rye1999"],"tags":["groes","kinetics","fret"],"difficulty":2,"url":"/machines/groel#evidence","cites":[{"source":"evidence:substrate-accelerates-groes-release","machine":"groel","label":"GroES release when non-native protein is present (Rye HS 1999)","section":"evidence","anchor":"ev-substrate-accelerates-groes-release"},{"source":"ref:rye1999","machine":"groel","label":"Rye et al.","section":"sources","anchor":"ref-rye1999","href":"https://doi.org/10.1016/s0092-8674(00)80742-4"}]},{"id":"groel-cage-volume-method","machine":"groel","kind":"qa","prompt":"How did Tang and colleagues (2006) change the volume of the GroEL cavity?","answer":"By deleting or extending the C-terminal Gly-Gly-Met repeats that hang into it.","explanation":"Shrinking the cavity by 1.9–4.4% sped folding of 33 kDa rhodanese and MetF, while any change slowed 41 kDa MBP and 50 kDa RuBisCo. The natural cage size suits fast folding, not just shelter.","section":"evidence","topic":"parts","sources":["evidence:cage-volume-sets-folding-speed","ref:tang2006"],"tags":["method","chamber"],"difficulty":3,"url":"/machines/groel#evidence","cites":[{"source":"evidence:cage-volume-sets-folding-speed","machine":"groel","label":"Effect of cavity volume on folding speed (Tang YC 2006)","section":"evidence","anchor":"ev-cage-volume-sets-folding-speed"},{"source":"ref:tang2006","machine":"groel","label":"Tang et al.","section":"sources","anchor":"ref-tang2006","href":"https://doi.org/10.1016/j.cell.2006.04.027"}]},{"id":"groel-sr1-folds-inside","machine":"groel","kind":"qa","prompt":"Rhodanese trapped under GroES in single-ring GroEL (SR1), which never opens, still became active. What does this show?","answer":"The protein folds inside the closed cage, not only after release.","explanation":"The trapped rhodanese reached native activity with a half-time of about 7 min while still bound to SR1.","section":"evidence","topic":"cycle","sources":["evidence:folding-inside-single-ring","ref:weissman1996"],"tags":["sr1","chamber"],"difficulty":2,"url":"/machines/groel#evidence","cites":[{"source":"evidence:folding-inside-single-ring","machine":"groel","label":"Folding inside a cage that never opens (Weissman JS 1996)","section":"evidence","anchor":"ev-folding-inside-single-ring"},{"source":"ref:weissman1996","machine":"groel","label":"Weissman et al.","section":"sources","anchor":"ref-weissman1996","href":"https://doi.org/10.1016/s0092-8674(00)81293-3"}]},{"id":"groel-one-or-two-lids-in-cells","machine":"groel","kind":"cloze","prompt":"In living E. coli cells, {{55 to 70%}} of GroEL carried one GroES lid and the rest carried two.","answer":"55 to 70%","explanation":"With client protein in vitro, Ye and Lorimer found most complexes carry GroES on both rings (\"footballs\"), so the simple one-lid alternating cycle is not the whole story.","section":"evidence","topic":"debate","sources":["step:The other ring takes its turn","ref:wagner2024","ref:ye2013"],"tags":["football","in-cell"],"difficulty":3,"url":"/machines/groel#evidence","cites":[{"source":"step:The other ring takes its turn","machine":"groel","label":"Step: The other ring takes its turn","section":"mechanism"},{"source":"ref:wagner2024","machine":"groel","label":"Wagner et al.","section":"sources","href":"https://doi.org/10.1038/s41586-024-07843-w"},{"source":"ref:ye2013","machine":"groel","label":"Ye and Lorimer, PNAS 2013","section":"sources","href":"https://doi.org/10.1073/pnas.1317702110"}]},{"id":"proteasome-tag-signal","machine":"proteasome","kind":"qa","prompt":"What mark does the cell put on a protein to send it to the 26S proteasome?","answer":"A chain of ubiquitin.","explanation":"Ubiquitin receptors in the proteasome bind this chain, so the proteasome picks marked proteins out of thousands of others.","section":"summary","topic":"purpose","sources":["machine:summary","step:A tagged protein arrives","ref:dong2019"],"tags":["ubiquitin","recognition"],"difficulty":1,"url":"/machines/proteasome#summary","cites":[{"source":"machine:summary","machine":"proteasome","label":"Summary","section":"summary"},{"source":"step:A tagged protein arrives","machine":"proteasome","label":"Step: A tagged protein arrives","section":"mechanism"},{"source":"ref:dong2019","machine":"proteasome","label":"Dong et al.","section":"sources","anchor":"ref-dong2019","href":"https://doi.org/10.1038/s41586-018-0736-4"}]},{"id":"proteasome-sealed-barrel-why","machine":"proteasome","kind":"qa","prompt":"Why does the proteasome keep its cutting sites inside a sealed barrel?","answer":"So that only a chain fed through the gate meets them, which keeps the cell's other proteins safe.","explanation":"All six active sites face the inner chamber, and the gate into it is shut until the ATPase ring opens it.","section":"summary","topic":"purpose","sources":["step:Cut into short peptides","stop:barrel","ref:groll1997"],"tags":["core-particle","safety"],"difficulty":2,"url":"/machines/proteasome#summary","cites":[{"source":"step:Cut into short peptides","machine":"proteasome","label":"Step: Cut into short peptides","section":"mechanism"},{"source":"stop:barrel","machine":"proteasome","label":"Big picture: Inside the barrel","section":"story"},{"source":"ref:groll1997","machine":"proteasome","label":"Groll et al.","section":"sources","anchor":"ref-groll1997","href":"https://doi.org/10.1038/386463a0"}]},{"id":"proteasome-atpase-ring-role","machine":"proteasome","kind":"qa","prompt":"Which part of the 26S proteasome grips, unfolds and pulls the tagged protein into the barrel?","answer":"The ring of six AAA+ ATPases, Rpt1 to Rpt6.","explanation":"The same ring also opens the gate into the core particle.","section":"summary","topic":"parts","sources":["component:Rpt1-Rpt6","stat:ATPase subunits","ref:lander2012"],"tags":["atpase","motor"],"difficulty":1,"url":"/machines/proteasome#summary","cites":[{"source":"component:Rpt1-Rpt6","machine":"proteasome","label":"Part: Rpt1-Rpt6","section":"summary"},{"source":"stat:ATPase subunits","machine":"proteasome","label":"Key number: ATPase subunits","section":"summary"},{"source":"ref:lander2012","machine":"proteasome","label":"Lander et al.","section":"sources","anchor":"ref-lander2012","href":"https://doi.org/10.1038/nature10774"}]},{"id":"proteasome-rpn11-role","machine":"proteasome","kind":"qa","prompt":"What does Rpn11, a zinc enzyme of the proteasome lid, do to the substrate?","answer":"It cuts the ubiquitin chain off the substrate at the pore entrance.","explanation":"Rpn11 is a deubiquitinase. It removes the tag in the same cycle that pulls the protein in.","section":"summary","topic":"parts","sources":["component:Rpn11","mechanism:Deubiquitination","ref:delapena2018"],"tags":["rpn11","ubiquitin"],"difficulty":1,"url":"/machines/proteasome#summary","cites":[{"source":"component:Rpn11","machine":"proteasome","label":"Part: Rpn11","section":"summary"},{"source":"mechanism:Deubiquitination","machine":"proteasome","label":"Step: Deubiquitination","section":"mechanism"},{"source":"ref:delapena2018","machine":"proteasome","label":"de la Pena et al.","section":"sources","anchor":"ref-delapena2018","href":"https://doi.org/10.1126/science.aav0725"}]},{"id":"proteasome-active-beta-subunits","machine":"proteasome","kind":"qa","prompt":"Which three beta subunits of the proteasome core particle carry the active sites?","answer":"beta1, beta2 and beta5.","explanation":"They cut after acidic, basic and bulky water-repelling residues: caspase-like, trypsin-like and chymotrypsin-like. The other four beta types are structural.","section":"summary","topic":"parts","sources":["component:beta1, beta2, beta5","step:Cut into short peptides","ref:groll1997"],"tags":["core-particle","active-site"],"difficulty":2,"url":"/machines/proteasome#summary","cites":[{"source":"component:beta1, beta2, beta5","machine":"proteasome","label":"Part: beta1, beta2, beta5","section":"summary"},{"source":"step:Cut into short peptides","machine":"proteasome","label":"Step: Cut into short peptides","section":"mechanism"},{"source":"ref:groll1997","machine":"proteasome","label":"Groll et al.","section":"sources","anchor":"ref-groll1997","href":"https://doi.org/10.1038/386463a0"}]},{"id":"proteasome-core-subunits","machine":"proteasome","kind":"cloze","prompt":"The proteasome core particle has 28 subunits in {{four rings of seven}}: (alpha1-7 beta1-7)2.","answer":"four rings of seven","explanation":"Two outer alpha rings form the gate; two inner beta rings hold the active sites, three per ring, so six sites per core.","section":"summary","topic":"numbers","sources":["stat:Core particle subunits","stat:Catalytic sites per core","ref:groll1997"],"tags":["core-particle","structure"],"difficulty":2,"url":"/machines/proteasome#summary","cites":[{"source":"stat:Core particle subunits","machine":"proteasome","label":"Key number: Core particle subunits","section":"summary"},{"source":"stat:Catalytic sites per core","machine":"proteasome","label":"Key number: Catalytic sites per core","section":"summary"},{"source":"ref:groll1997","machine":"proteasome","label":"Groll et al.","section":"sources","anchor":"ref-groll1997","href":"https://doi.org/10.1038/386463a0"}]},{"id":"proteasome-hbyx-older","machine":"proteasome","kind":"qa","prompt":"Why is the proteasome's HbYX gate signal thought to be older than the 26S particle itself?","answer":"Archaea use the simpler PAN ATPase with the same HbYX gate signal as the eukaryotic Rpt subunits.","explanation":"Free HbYX peptides of 7 to 10 residues open the gate of the archaeal core on their own (Smith et al. 2007).","section":"summary","topic":"parts","sources":["evolution:AAA+ origin of the ATPase ring","ref:smith2007","ref:rabl2008"],"tags":["evolution","gate"],"difficulty":3,"url":"/machines/proteasome#summary","cites":[{"source":"evolution:AAA+ origin of the ATPase ring","machine":"proteasome","label":"AAA+ origin of the ATPase ring","section":"summary"},{"source":"ref:smith2007","machine":"proteasome","label":"Smith et al.","section":"sources","anchor":"ref-smith2007","href":"https://doi.org/10.1016/j.molcel.2007.06.033"},{"source":"ref:rabl2008","machine":"proteasome","label":"Rabl et al.","section":"sources","anchor":"ref-rabl2008","href":"https://doi.org/10.1016/j.molcel.2008.03.004"}]},{"id":"proteasome-protac","machine":"proteasome","kind":"qa","prompt":"How does a PROTAC get the proteasome to destroy a chosen protein?","answer":"It ties the protein to an E3 ligase, which marks it with ubiquitin.","explanation":"The two-headed molecule supplies the address; the proteasome does the rest. dBET1 removed BET proteins in cells and in mice (status: demonstrated).","section":"summary","topic":"debate","sources":["frontier:Targeted degradation with PROTACs","ref:winter2015","ref:sakamoto2001"],"tags":["protac","drugs"],"difficulty":2,"url":"/machines/proteasome#summary","cites":[{"source":"frontier:Targeted degradation with PROTACs","machine":"proteasome","label":"Open question: Targeted degradation with PROTACs","section":"summary"},{"source":"ref:winter2015","machine":"proteasome","label":"Winter et al.","section":"sources","anchor":"ref-winter2015","href":"https://doi.org/10.1126/science.aab1433"},{"source":"ref:sakamoto2001","machine":"proteasome","label":"Sakamoto et al.","section":"sources","anchor":"ref-sakamoto2001","href":"https://doi.org/10.1073/pnas.141230798"}]},{"id":"proteasome-commitment","machine":"proteasome","kind":"qa","prompt":"Ubiquitin binding alone does not commit a protein to the proteasome. What does?","answer":"The pore loops of the ATPase ring gripping the protein's unstructured tail.","explanation":"Ubiquitin chains bind and leave quickly. Only a substrate whose tail engages the motor triggers the switch to the working shape and goes on to be degraded.","section":"mechanism","topic":"cycle","sources":["step:The loose tail enters the pore","step:A tagged protein arrives","ref:bard2019"],"tags":["engagement","tail"],"difficulty":2,"url":"/machines/proteasome#mechanism","cites":[{"source":"step:The loose tail enters the pore","machine":"proteasome","label":"Step: The loose tail enters the pore","section":"mechanism"},{"source":"step:A tagged protein arrives","machine":"proteasome","label":"Step: A tagged protein arrives","section":"mechanism"},{"source":"ref:bard2019","machine":"proteasome","label":"Bard et al.","section":"sources","anchor":"ref-bard2019","href":"https://doi.org/10.1016/j.cell.2019.02.031"}]},{"id":"proteasome-pull-speeds-cut","machine":"proteasome","kind":"cloze","prompt":"Rpn11 cuts the ubiquitin chain off faster when {{the motor pulls on the substrate}}.","answer":"the motor pulls on the substrate","explanation":"This couples tag removal to translocation: the tag comes off as the protein moves into the pore.","section":"mechanism","topic":"cycle","sources":["mechanism:Deubiquitination","step:Rpn11 cuts off the tag","ref:delapena2018"],"tags":["rpn11","coupling"],"difficulty":2,"url":"/machines/proteasome#mechanism","cites":[{"source":"mechanism:Deubiquitination","machine":"proteasome","label":"Step: Deubiquitination","section":"mechanism"},{"source":"step:Rpn11 cuts off the tag","machine":"proteasome","label":"Step: Rpn11 cuts off the tag","section":"mechanism"},{"source":"ref:delapena2018","machine":"proteasome","label":"de la Pena et al.","section":"sources","anchor":"ref-delapena2018","href":"https://doi.org/10.1126/science.aav0725"}]},{"id":"proteasome-gate-opening","machine":"proteasome","kind":"qa","prompt":"How do the proteasome's ATPases open the gate into the core particle?","answer":"Their C-terminal HbYX tails dock in pockets between alpha subunits, which rotates the alpha subunits and opens the gate.","explanation":"The tails work like a key in a lock. Closed, the N-terminal tails of the alpha subunits fill the entrance.","section":"mechanism","topic":"cycle","sources":["mechanism:Gate opening","step:The gate opens","ref:smith2007"],"tags":["gate","hbyx"],"difficulty":2,"url":"/machines/proteasome#mechanism","cites":[{"source":"mechanism:Gate opening","machine":"proteasome","label":"Step: Gate opening","section":"mechanism"},{"source":"step:The gate opens","machine":"proteasome","label":"Step: The gate opens","section":"mechanism"},{"source":"ref:smith2007","machine":"proteasome","label":"Smith et al.","section":"sources","anchor":"ref-smith2007","href":"https://doi.org/10.1016/j.molcel.2007.06.033"}]},{"id":"proteasome-staircase-one-way","machine":"proteasome","kind":"qa","prompt":"The six proteasome ATPases stand in a spiral staircase. Why does the substrate chain move one way only?","answer":"ATP hydrolysis passes around the ring in order, so each subunit grips the chain in turn.","explanation":"The gripping subunits move down together and carry the chain with them, hand over hand.","section":"mechanism","topic":"cycle","sources":["mechanism:Translocation in steps","step:Hand over hand","ref:dong2019"],"tags":["atpase","translocation"],"difficulty":2,"url":"/machines/proteasome#mechanism","cites":[{"source":"mechanism:Translocation in steps","machine":"proteasome","label":"Step: Translocation in steps","section":"mechanism"},{"source":"step:Hand over hand","machine":"proteasome","label":"Step: Hand over hand","section":"mechanism"},{"source":"ref:dong2019","machine":"proteasome","label":"Dong et al.","section":"sources","anchor":"ref-dong2019","href":"https://doi.org/10.1038/s41586-018-0736-4"}]},{"id":"proteasome-seam-subunit","machine":"proteasome","kind":"qa","prompt":"In the proteasome's ATPase staircase, what does the subunit at the bottom do next?","answer":"It lets go of the chain and climbs to the top.","explanation":"This 'seam' subunit then binds ATP and grips the chain again at the top, so the ring keeps cycling.","section":"mechanism","topic":"cycle","sources":["step:Hand over hand","evidence:substrate-engaged-motor-states","ref:delapena2018"],"tags":["atpase","translocation"],"difficulty":2,"url":"/machines/proteasome#mechanism","cites":[{"source":"step:Hand over hand","machine":"proteasome","label":"Step: Hand over hand","section":"mechanism"},{"source":"evidence:substrate-engaged-motor-states","machine":"proteasome","label":"Motor states seen in the substrate-engaged yeast 26S proteasome (de la Peña AH 2018)","section":"evidence","anchor":"ev-substrate-engaged-motor-states"},{"source":"ref:delapena2018","machine":"proteasome","label":"de la Pena et al.","section":"sources","anchor":"ref-delapena2018","href":"https://doi.org/10.1126/science.aav0725"}]},{"id":"proteasome-hinge","machine":"proteasome","kind":"cloze","prompt":"In each proteasome ATPase, a {{hinge motion}} driven by ATP hydrolysis sets when that subunit holds or releases the chain.","answer":"hinge motion","explanation":"Bound nucleotide locks the large and small AAA+ subdomains into one rigid body; release lets them hinge by 20–25°.","section":"mechanism","topic":"cycle","sources":["mechanism:Unfolding","step:Hand over hand","ref:dong2019"],"tags":["atpase","unfolding"],"difficulty":3,"url":"/machines/proteasome#mechanism","cites":[{"source":"mechanism:Unfolding","machine":"proteasome","label":"Step: Unfolding","section":"mechanism"},{"source":"step:Hand over hand","machine":"proteasome","label":"Step: Hand over hand","section":"mechanism"},{"source":"ref:dong2019","machine":"proteasome","label":"Dong et al.","section":"sources","anchor":"ref-dong2019","href":"https://doi.org/10.1038/s41586-018-0736-4"}]},{"id":"proteasome-thr1","machine":"proteasome","kind":"qa","prompt":"Which residue in the proteasome's active beta subunits attacks the peptide bond?","answer":"The N-terminal threonine (Thr1).","explanation":"It sits on beta1, beta2 and beta5. Deleting Thr1 or changing it to alanine stops the enzyme.","section":"mechanism","topic":"cycle","sources":["mechanism:Cutting","stat:Catalytic residue","ref:seemuller1995"],"tags":["active-site","threonine"],"difficulty":1,"url":"/machines/proteasome#mechanism","cites":[{"source":"mechanism:Cutting","machine":"proteasome","label":"Step: Cutting","section":"mechanism"},{"source":"stat:Catalytic residue","machine":"proteasome","label":"Key number: Catalytic residue","section":"summary"},{"source":"ref:seemuller1995","machine":"proteasome","label":"Seemueller et al.","section":"sources","anchor":"ref-seemuller1995","href":"https://doi.org/10.1126/science.7725107"}]},{"id":"proteasome-release","machine":"proteasome","kind":"qa","prompt":"What decides when peptides leave the proteasome barrel, according to Kisselev and colleagues' proposal?","answer":"Cutting goes on until a piece is short enough to diffuse out.","explanation":"Product length hardly changed when one type of active site was blocked. The products are 3 to 22 residues long.","section":"mechanism","topic":"cycle","sources":["mechanism:Release","step:Cut into short peptides","ref:kisselev1999"],"tags":["peptides","release"],"difficulty":2,"url":"/machines/proteasome#mechanism","cites":[{"source":"mechanism:Release","machine":"proteasome","label":"Step: Release","section":"mechanism"},{"source":"step:Cut into short peptides","machine":"proteasome","label":"Step: Cut into short peptides","section":"mechanism"},{"source":"ref:kisselev1999","machine":"proteasome","label":"Kisselev et al.","section":"sources","anchor":"ref-kisselev1999","href":"https://doi.org/10.1074/jbc.274.6.3363"}]},{"id":"proteasome-body-turnover","machine":"proteasome","kind":"cloze","prompt":"An adult human breaks down and rebuilds about {{300–400 g}} of body protein a day, but eats only 50–80 g.","answer":"300–400 g","explanation":"Most of the parts are recycled, and most of that breakdown happens in the proteasome. The figure is a review estimate.","section":"story","topic":"purpose","sources":["fact:P1","stop:body","ref:schutz2011"],"tags":["body","turnover"],"difficulty":2,"url":"/machines/proteasome#story","cites":[{"source":"fact:P1","machine":"proteasome","label":"Protein broken down and rebuilt per day: 300–400 g per day","section":"story"},{"source":"stop:body","machine":"proteasome","label":"Big picture: You, renewing","section":"story"},{"source":"ref:schutz2011","machine":"proteasome","label":"Schutz 2011","section":"sources","href":"https://doi.org/10.1024/0300-9831/a000064"}]},{"id":"proteasome-pieces-fate","machine":"proteasome","kind":"qa","prompt":"Most proteasome peptides become amino acids for new proteins. What happens to a few of them?","answer":"They go to the cell surface on MHC class I, where immune cells check them.","explanation":"The display is a sample of what the cell is making, so immune cells can spot an infected cell.","section":"story","topic":"purpose","sources":["stop:pieces","fact:P8-mhc","ref:schubert2000"],"tags":["immune","mhc"],"difficulty":1,"url":"/machines/proteasome#story","cites":[{"source":"stop:pieces","machine":"proteasome","label":"Big picture: The pieces","section":"story"},{"source":"fact:P8-mhc","machine":"proteasome","label":"Length of the peptides shown on MHC class I: 8–10 amino acids","section":"story"},{"source":"ref:schubert2000","machine":"proteasome","label":"Schubert 2000","section":"sources","href":"https://doi.org/10.1038/35008096"}]},{"id":"proteasome-vs-ribosome-rate","machine":"proteasome","kind":"qa","prompt":"In mouse L929 cells, how does the number of proteins the proteasomes destroy per minute compare with the number the ribosomes make?","answer":"About half: 2 × 10^6 destroyed versus 4 × 10^6 made per minute.","explanation":"It is a calculation: 8 × 10^5 proteasomes × 2.5 proteins per minute each. Together the two machines set protein levels.","section":"story","topic":"numbers","sources":["fact:P12","fact:P11","link:ribosome","ref:princiotta2003"],"tags":["cell","ribosome"],"difficulty":3,"url":"/machines/proteasome#story","cites":[{"source":"fact:P12","machine":"proteasome","label":"Proteins destroyed per minute in one mouse cell: 2 × 10^6 per minute","section":"story"},{"source":"fact:P11","machine":"proteasome","label":"Proteasomes in one mouse cell: 8 × 10^5 proteasomes","section":"story"},{"source":"link:ribosome","machine":"proteasome","label":"Link to Ribosome","section":"story"},{"source":"ref:princiotta2003","machine":"proteasome","label":"Princiotta 2003","section":"sources","href":"https://doi.org/10.1016/s1074-7613(03)00051-7"}]},{"id":"proteasome-shredder-breaks","machine":"proteasome","kind":"qa","prompt":"Where does the analogy 'the proteasome is a paper shredder' break down about how input gets in?","answer":"A shredder is fed; the proteasome pulls and unfolds its input itself, with six ATP motors.","explanation":"It also takes off the ubiquitin tag and returns it for reuse. The analogy gets right that the enclosed blades hurt nothing else.","section":"story","topic":"purpose","sources":["analogy:A paper shredder with a doorman"],"tags":["analogy"],"difficulty":2,"url":"/machines/proteasome#story","cites":[{"source":"analogy:A paper shredder with a doorman","machine":"proteasome","label":"Analogy: A paper shredder with a doorman","section":"story"}]},{"id":"proteasome-healthy-proteins","machine":"proteasome","kind":"qa","prompt":"Unlike a recycling plant, the proteasome also destroys healthy proteins on schedule. What does that let the cell do?","answer":"Turn signals off.","explanation":"For example, the proteasome destroys transcription factors, so it helps decide when genes switch on.","section":"story","topic":"purpose","sources":["analogy:A recycling plant","link:rna-polymerase"],"tags":["analogy","signalling"],"difficulty":2,"url":"/machines/proteasome#story","cites":[{"source":"analogy:A recycling plant","machine":"proteasome","label":"Analogy: A recycling plant","section":"story"},{"source":"link:rna-polymerase","machine":"proteasome","label":"Link to RNA polymerase II","section":"story"}]},{"id":"proteasome-time-per-protein","machine":"proteasome","kind":"cloze","prompt":"One mouse 26S proteasome needs about {{13 s}} to degrade one ubiquitinated DHFR molecule (Ub5-DHFR).","answer":"13 s","explanation":"A longer ubiquitinated protein, Sic1, took about twice as long (26 s), so time depends on the substrate.","section":"evidence","topic":"numbers","sources":["evidence:degradation-time-ub5-dhfr","stat:Time to degrade one tagged protein","ref:peth2013"],"tags":["rate","dhfr"],"difficulty":2,"url":"/machines/proteasome#evidence","cites":[{"source":"evidence:degradation-time-ub5-dhfr","machine":"proteasome","label":"Time for one 26S proteasome to degrade one ubiquitinated DHFR molecule (Peth A 2013)","section":"evidence","anchor":"ev-degradation-time-ub5-dhfr"},{"source":"stat:Time to degrade one tagged protein","machine":"proteasome","label":"Key number: Time to degrade one tagged protein","section":"summary"},{"source":"ref:peth2013","machine":"proteasome","label":"Peth et al.","section":"sources","anchor":"ref-peth2013","href":"https://doi.org/10.1074/jbc.m113.482570"}]},{"id":"proteasome-time-method","machine":"proteasome","kind":"qa","prompt":"How did Peth and colleagues get the 13 s a proteasome needs per Ub5-DHFR molecule?","answer":"From the maximal degradation rate (Vmax) at rising substrate levels: time per molecule = 1/Vmax.","explanation":"They followed 32P-labelled substrate turning into acid-soluble peptides with purified mouse 26S proteasomes; the maximum was about 4.7 molecules per minute per particle.","section":"evidence","topic":"numbers","sources":["evidence:degradation-time-ub5-dhfr","ref:peth2013"],"tags":["method","kinetics"],"difficulty":3,"url":"/machines/proteasome#evidence","cites":[{"source":"evidence:degradation-time-ub5-dhfr","machine":"proteasome","label":"Time for one 26S proteasome to degrade one ubiquitinated DHFR molecule (Peth A 2013)","section":"evidence","anchor":"ev-degradation-time-ub5-dhfr"},{"source":"ref:peth2013","machine":"proteasome","label":"Peth et al.","section":"sources","anchor":"ref-peth2013","href":"https://doi.org/10.1074/jbc.m113.482570"}]},{"id":"proteasome-atp-cost","machine":"proteasome","kind":"cloze","prompt":"Mouse 26S proteasomes spend {{50–80}} ATP to destroy one Ub5-DHFR molecule.","answer":"50–80","explanation":"Peth and colleagues combined the maximal degradation rate with ATP hydrolysis measured by a malachite green phosphate assay.","section":"evidence","topic":"numbers","sources":["evidence:atp-cost-ub5-dhfr","stat:ATP cost per tagged protein","fact:P9","ref:peth2013"],"tags":["atp","cost"],"difficulty":2,"url":"/machines/proteasome#evidence","cites":[{"source":"evidence:atp-cost-ub5-dhfr","machine":"proteasome","label":"ATP hydrolysed per ubiquitinated DHFR molecule degraded (Peth A 2013)","section":"evidence","anchor":"ev-atp-cost-ub5-dhfr"},{"source":"stat:ATP cost per tagged protein","machine":"proteasome","label":"Key number: ATP cost per tagged protein","section":"summary"},{"source":"fact:P9","machine":"proteasome","label":"ATP spent to destroy one tagged protein: 50–80 ATP","section":"story"},{"source":"ref:peth2013","machine":"proteasome","label":"Peth et al.","section":"sources","anchor":"ref-peth2013","href":"https://doi.org/10.1074/jbc.m113.482570"}]},{"id":"proteasome-folate-meaning","machine":"proteasome","kind":"qa","prompt":"Folic acid stabilises the DHFR fold. What happened to proteasome ATP use per minute and to degradation time when it was bound?","answer":"ATP use per minute stayed the same, but degradation slowed from 13 s to 23 s per molecule.","explanation":"So each molecule cost more, 90–140 ATP instead of 50–80: unfolding is a slow and costly step for a stable protein.","section":"evidence","topic":"numbers","sources":["evidence:folate-slows-degradation","step:The fold is pulled apart","ref:peth2013"],"tags":["unfolding","atp"],"difficulty":3,"url":"/machines/proteasome#evidence","cites":[{"source":"evidence:folate-slows-degradation","machine":"proteasome","label":"Effect of stabilising the DHFR fold with folic acid (Peth A 2013)","section":"evidence","anchor":"ev-folate-slows-degradation"},{"source":"step:The fold is pulled apart","machine":"proteasome","label":"Step: The fold is pulled apart","section":"mechanism"},{"source":"ref:peth2013","machine":"proteasome","label":"Peth et al.","section":"sources","anchor":"ref-peth2013","href":"https://doi.org/10.1074/jbc.m113.482570"}]},{"id":"proteasome-single-rpt-mutant","machine":"proteasome","kind":"qa","prompt":"Blocking ATP binding in one yeast proteasome ATPase (Rpt3, Rpt5 or Rpt6) cut basal ATP use by about 66%, not the 16% expected. What does that show?","answer":"The six ATPases run in an ordered cycle, so one stalled subunit holds up the others.","explanation":"If the subunits worked on their own, losing one of six would cost only about one sixth of the activity.","section":"evidence","topic":"cycle","sources":["evidence:rpt-single-mutant-atpase","step:Hand over hand","ref:peth2013"],"tags":["atpase","coordination"],"difficulty":3,"url":"/machines/proteasome#evidence","cites":[{"source":"evidence:rpt-single-mutant-atpase","machine":"proteasome","label":"Loss of basal ATPase activity when one Rpt subunit cannot bind ATP (Peth A 2013)","section":"evidence","anchor":"ev-rpt-single-mutant-atpase"},{"source":"step:Hand over hand","machine":"proteasome","label":"Step: Hand over hand","section":"mechanism"},{"source":"ref:peth2013","machine":"proteasome","label":"Peth et al.","section":"sources","anchor":"ref-peth2013","href":"https://doi.org/10.1074/jbc.m113.482570"}]},{"id":"proteasome-step-size","machine":"proteasome","kind":"qa","prompt":"In cryo-EM of working yeast 26S proteasomes, how far does the substrate move per ATP?","answer":"About 6 Å, read as two amino acids per ATP.","explanation":"De la Peña and colleagues saw the engaged pore-1 loop tyrosines move down by that distance between consecutive motor states.","section":"evidence","topic":"numbers","sources":["evidence:translocation-step-size","step:Hand over hand","ref:delapena2018"],"tags":["translocation","cryo-em"],"difficulty":2,"url":"/machines/proteasome#evidence","cites":[{"source":"evidence:translocation-step-size","machine":"proteasome","label":"Substrate translocation step per hydrolysed ATP (de la Peña AH 2018)","section":"evidence","anchor":"ev-translocation-step-size"},{"source":"step:Hand over hand","machine":"proteasome","label":"Step: Hand over hand","section":"mechanism"},{"source":"ref:delapena2018","machine":"proteasome","label":"de la Pena et al.","section":"sources","anchor":"ref-delapena2018","href":"https://doi.org/10.1126/science.aav0725"}]},{"id":"proteasome-mass-not-measured","machine":"proteasome","kind":"qa","prompt":"The human 26S proteasome is often given as 2.5 MDa, citing Dong et al. 2019. What is the problem with that source?","answer":"Dong et al. state the mass but do not measure it; a primary measurement still has to be found.","explanation":"Their paper is a cryo-EM structure study, and its own results are the seven structures, not a mass.","section":"evidence","topic":"debate","sources":["evidence:holoenzyme-mass","stat:Mass of the holoenzyme","ref:dong2019"],"tags":["mass","provenance"],"difficulty":2,"url":"/machines/proteasome#evidence","cites":[{"source":"evidence:holoenzyme-mass","machine":"proteasome","label":"Mass of the human 26S proteasome holoenzyme (Dong Y 2019)","section":"evidence","anchor":"ev-holoenzyme-mass"},{"source":"stat:Mass of the holoenzyme","machine":"proteasome","label":"Key number: Mass of the holoenzyme","section":"summary"},{"source":"ref:dong2019","machine":"proteasome","label":"Dong et al.","section":"sources","anchor":"ref-dong2019","href":"https://doi.org/10.1038/s41586-018-0736-4"}]},{"id":"serca-job","machine":"serca","kind":"qa","prompt":"What job does SERCA do in a muscle cell after each contraction?","answer":"It clears calcium from the cytoplasm, pumping it back into the sarcoplasmic reticulum.","explanation":"Calcium switches muscle on, so the muscle can only relax and fire again once SERCA has taken the calcium away.","section":"summary","topic":"purpose","sources":["machine:summary","stop:twitch"],"tags":["calcium","muscle"],"difficulty":1,"url":"/machines/serca#summary","cites":[{"source":"machine:summary","machine":"serca","label":"Summary","section":"summary"},{"source":"stop:twitch","machine":"serca","label":"Big picture: One twitch","section":"story"}]},{"id":"serca-ca-per-atp","machine":"serca","kind":"cloze","prompt":"SERCA burns one ATP to push {{two}} calcium ions out of the cytoplasm.","answer":"two","explanation":"The crystal structure shows two calcium sites side by side inside the membrane domain, which fits this ratio.","section":"summary","topic":"numbers","sources":["stat:Calcium ions per ATP","machine:tagline","ref:toyoshima2000"],"tags":["stoichiometry","atp"],"difficulty":1,"url":"/machines/serca#summary","cites":[{"source":"stat:Calcium ions per ATP","machine":"serca","label":"Key number: Calcium ions per ATP","section":"summary"},{"source":"machine:tagline","machine":"serca","label":"Summary","section":"summary"},{"source":"ref:toyoshima2000","machine":"serca","label":"Toyoshima et al.","section":"sources","anchor":"ref-toyoshima2000","href":"https://doi.org/10.1038/35015017"}]},{"id":"serca-p-domain","machine":"serca","kind":"qa","prompt":"Which SERCA domain holds the aspartate that accepts the phosphoryl group from ATP?","answer":"The P (phosphorylation) domain.","explanation":"The P domain has the same fold as haloacid dehalogenase, a general phosphotransfer scaffold.","section":"summary","topic":"parts","sources":["component:P domain","evolution:A borrowed catalytic core","ref:toyoshima2000"],"tags":["domains","phosphorylation"],"difficulty":1,"url":"/machines/serca#summary","cites":[{"source":"component:P domain","machine":"serca","label":"Part: P domain","section":"summary"},{"source":"evolution:A borrowed catalytic core","machine":"serca","label":"A borrowed catalytic core","section":"summary"},{"source":"ref:toyoshima2000","machine":"serca","label":"Toyoshima et al.","section":"sources","anchor":"ref-toyoshima2000","href":"https://doi.org/10.1038/35015017"}]},{"id":"serca-n-domain","machine":"serca","kind":"qa","prompt":"What part of ATP does SERCA's N domain bind?","answer":"The adenosine part.","explanation":"When nucleotide binds, the N domain swings onto the P domain, which holds the aspartate that takes the phosphate.","section":"summary","topic":"parts","sources":["component:N domain","evidence:three-cytoplasmic-domains"],"tags":["domains","atp"],"difficulty":2,"url":"/machines/serca#summary","cites":[{"source":"component:N domain","machine":"serca","label":"Part: N domain","section":"summary"},{"source":"evidence:three-cytoplasmic-domains","machine":"serca","label":"Cytoplasmic domains of SERCA1a (Toyoshima C 2000)","section":"evidence","anchor":"ev-three-cytoplasmic-domains"}]},{"id":"serca-a-domain","machine":"serca","kind":"qa","prompt":"What does SERCA's A (actuator) domain drive when it tilts against the membrane helices?","answer":"Gating and dephosphorylation.","explanation":"Release of ADP opens the lumenal gate and release of phosphate closes it, mainly through movements of the A domain.","section":"summary","topic":"parts","sources":["component:A domain","evidence:four-principal-states"],"tags":["domains","gating"],"difficulty":2,"url":"/machines/serca#summary","cites":[{"source":"component:A domain","machine":"serca","label":"Part: A domain","section":"summary"},{"source":"evidence:four-principal-states","machine":"serca","label":"Reaction-cycle states with atomic models (Toyoshima C 2004)","section":"evidence","anchor":"ev-four-principal-states"}]},{"id":"serca-phospholamban-role","machine":"serca","kind":"qa","prompt":"How does phospholamban change the cardiac SERCA pump?","answer":"It raises the calcium level the pump needs to cycle.","explanation":"Phospholamban is an inhibitory membrane micropeptide of cardiac muscle: the same pump rate then needs more calcium.","section":"summary","topic":"parts","sources":["component:phospholamban","ref:maclennan2003"],"tags":["regulation","heart"],"difficulty":2,"url":"/machines/serca#summary","cites":[{"source":"component:phospholamban","machine":"serca","label":"Part: phospholamban","section":"summary"},{"source":"ref:maclennan2003","machine":"serca","label":"MacLennan and Kranias, Nat Rev Mol Cell Biol 2003","section":"sources","anchor":"ref-maclennan2003","href":"https://doi.org/10.1038/nrm1151"}]},{"id":"serca-dworf-safer","machine":"serca","kind":"qa","prompt":"Why is the activator DWORF seen as a safer way to boost SERCA than removing phospholamban?","answer":"Complete loss of phospholamban is lethal in humans; DWORF instead raises turnover and competes phospholamban off its site.","explanation":"This is a lab-scale idea: DWORF is the only known activator in the regulin family.","section":"summary","topic":"debate","sources":["frontier:Activator instead of brake removal","ref:verry2026"],"tags":["heart-failure","regulation"],"difficulty":2,"url":"/machines/serca#summary","cites":[{"source":"frontier:Activator instead of brake removal","machine":"serca","label":"Open question: Activator instead of brake removal","section":"summary"},{"source":"ref:verry2026","machine":"serca","label":"Verry and Makarewich, Front Cell Dev Biol 2026","section":"sources","anchor":"ref-verry2026","href":"https://doi.org/10.3389/fcell.2026.1864847"}]},{"id":"serca-gene-transfer-status","machine":"serca","kind":"qa","prompt":"What is the state of SERCA2a gene transfer as a treatment for heart failure?","answer":"It restored contractile function in failing heart muscle, but clinical results have been inconsistent.","explanation":"SR calcium cycling and SERCA2a activity fall in heart failure. The approach is still rated lab-scale.","section":"summary","topic":"debate","sources":["frontier:Gene transfer for heart failure","ref:periasamy2007"],"tags":["heart-failure"],"difficulty":2,"url":"/machines/serca#summary","cites":[{"source":"frontier:Gene transfer for heart failure","machine":"serca","label":"Open question: Gene transfer for heart failure","section":"summary"},{"source":"ref:periasamy2007","machine":"serca","label":"Periasamy and Kalyanasundaram, Muscle Nerve 2007","section":"sources","anchor":"ref-periasamy2007","href":"https://doi.org/10.1002/mus.20745"}]},{"id":"serca-e1-magnesium","machine":"serca","kind":"qa","prompt":"In SERCA's calcium-free E1 state, one Mg2+ sits in a calcium site. What does it block until calcium arrives?","answer":"Phosphorylation.","explanation":"Calcium is the trigger: once the two high-affinity sites fill, phosphorylation can go ahead. In skeletal muscle, sarcolipin stabilises this E1 state with magnesium.","section":"mechanism","topic":"cycle","sources":["mechanism:E1 waits with magnesium","component:sarcolipin","ref:toyoshima2013"],"tags":["e1","magnesium"],"difficulty":2,"url":"/machines/serca#mechanism","cites":[{"source":"mechanism:E1 waits with magnesium","machine":"serca","label":"Step: E1 waits with magnesium","section":"mechanism"},{"source":"component:sarcolipin","machine":"serca","label":"Part: sarcolipin","section":"summary"},{"source":"ref:toyoshima2013","machine":"serca","label":"Toyoshima et al.","section":"sources","anchor":"ref-toyoshima2013","href":"https://doi.org/10.1038/nature11899"}]},{"id":"serca-atp-bridges","machine":"serca","kind":"qa","prompt":"What does ATP binding do to SERCA's N and P domains?","answer":"It bridges them, pulling the widely separated domains together.","explanation":"The A domain then tilts and one membrane helix moves to lock the cytoplasmic gate, occluding the two calcium ions.","section":"mechanism","topic":"cycle","sources":["mechanism:ATP bridges the N and P domains","ref:toyoshima2004atp"],"tags":["atp","domains"],"difficulty":2,"url":"/machines/serca#mechanism","cites":[{"source":"mechanism:ATP bridges the N and P domains","machine":"serca","label":"Step: ATP bridges the N and P domains","section":"mechanism"},{"source":"ref:toyoshima2004atp","machine":"serca","label":"Toyoshima et al.","section":"sources","anchor":"ref-toyoshima2004atp","href":"https://doi.org/10.1038/nature02680"}]},{"id":"serca-no-backflow","machine":"serca","kind":"qa","prompt":"When SERCA phosphorylates itself, what stops the bound calcium from flowing back to the cytoplasm?","answer":"Helices M1 and M2 shift and close the cytosolic entrance.","explanation":"The same movement that transfers the phosphate to the aspartate shuts the entry door, so the ions are trapped before the exit opens.","section":"mechanism","topic":"cycle","sources":["mechanism:The pump phosphorylates itself","ref:sorensen2004"],"tags":["gating","phosphorylation"],"difficulty":2,"url":"/machines/serca#mechanism","cites":[{"source":"mechanism:The pump phosphorylates itself","machine":"serca","label":"Step: The pump phosphorylates itself","section":"mechanism"},{"source":"ref:sorensen2004","machine":"serca","label":"Sorensen et al.","section":"sources","anchor":"ref-sorensen2004","href":"https://doi.org/10.1126/science.1099366"}]},{"id":"serca-lumenal-gate-trigger","machine":"serca","kind":"cloze","prompt":"In SERCA, the change from E1P to E2P after {{phosphorylation}} opens the exit path for calcium to the lumen.","answer":"phosphorylation","explanation":"Helices M1 to M6 open the path, and a shift of M4 exposes Glu 309, Glu 771 and Asn 796 to the lumen, where calcium leaves.","section":"mechanism","topic":"cycle","sources":["mechanism:The lumenal gate opens and calcium leaves","ref:olesen2007"],"tags":["gating","e2p"],"difficulty":2,"url":"/machines/serca#mechanism","cites":[{"source":"mechanism:The lumenal gate opens and calcium leaves","machine":"serca","label":"Step: The lumenal gate opens and calcium leaves","section":"mechanism"},{"source":"ref:olesen2007","machine":"serca","label":"Olesen et al.","section":"sources","anchor":"ref-olesen2007","href":"https://doi.org/10.1038/nature06418"}]},{"id":"serca-protons-empty-sites","machine":"serca","kind":"qa","prompt":"After SERCA releases its two calcium ions to the lumen, what binds the empty sites?","answer":"Protons, which become occluded.","explanation":"The pump carries protons back the other way; then the phosphoenzyme is hydrolysed and the cycle can reset.","section":"mechanism","topic":"cycle","sources":["mechanism:Protons take the empty seats","ref:olesen2004"],"tags":["protons","counter-transport"],"difficulty":2,"url":"/machines/serca#mechanism","cites":[{"source":"mechanism:Protons take the empty seats","machine":"serca","label":"Step: Protons take the empty seats","section":"mechanism"},{"source":"ref:olesen2004","machine":"serca","label":"Olesen et al.","section":"sources","anchor":"ref-olesen2004","href":"https://doi.org/10.1126/science.1106289"}]},{"id":"serca-tges-motif","machine":"serca","kind":"cloze","prompt":"SERCA's phosphoenzyme is hydrolysed through the conserved {{Thr-Gly-Glu-Ser}} motif, by the same associative chemistry as the forward transfer.","answer":"Thr-Gly-Glu-Ser","explanation":"This motif (TGES) lets water attack the phosphate while the counter-transported protons stay occluded.","section":"mechanism","topic":"cycle","sources":["mechanism:Protons take the empty seats","evidence:counterion-occlusion","ref:olesen2004"],"tags":["dephosphorylation"],"difficulty":3,"url":"/machines/serca#mechanism","cites":[{"source":"mechanism:Protons take the empty seats","machine":"serca","label":"Step: Protons take the empty seats","section":"mechanism"},{"source":"evidence:counterion-occlusion","machine":"serca","label":"State in which the counter-transported protons are occluded (Olesen C 2004)","section":"evidence","anchor":"ev-counterion-occlusion"},{"source":"ref:olesen2004","machine":"serca","label":"Olesen et al.","section":"sources","anchor":"ref-olesen2004","href":"https://doi.org/10.1126/science.1106289"}]},{"id":"serca-e2-headpiece","machine":"serca","kind":"cloze","prompt":"In SERCA's calcium-free E2 state, the three cytoplasmic domains gather into {{one compact headpiece}}.","answer":"one compact headpiece","explanation":"Without nucleotide in E1 they sit far apart; between the states, six of the ten membrane helices rearrange.","section":"mechanism","topic":"cycle","sources":["mechanism:E2 relaxes back to E1","evidence:e2-thapsigargin-structure","ref:toyoshima2002"],"tags":["e2","domains"],"difficulty":2,"url":"/machines/serca#mechanism","cites":[{"source":"mechanism:E2 relaxes back to E1","machine":"serca","label":"Step: E2 relaxes back to E1","section":"mechanism"},{"source":"evidence:e2-thapsigargin-structure","machine":"serca","label":"Changes from the Ca2+-bound E1 state to the Ca2+-free E2 state (Toyoshima C 2002)","section":"evidence","anchor":"ev-e2-thapsigargin-structure"},{"source":"ref:toyoshima2002","machine":"serca","label":"Toyoshima and Nomura, Nature 2002","section":"sources","anchor":"ref-toyoshima2002","href":"https://doi.org/10.1038/nature00944"}]},{"id":"serca-gradient-size","machine":"serca","kind":"cloze","prompt":"At rest, the SERCA pumps hold about {{4,000}} times more free calcium inside the sarcoplasmic reticulum than in the muscle cytosol.","answer":"4,000","explanation":"Calculated from free Ca2+ of about 390 µM in the SR and about 100 nM in the cytosol of mouse fibres. The often-quoted 10,000-fold is outside the cell versus the cytosol.","section":"story","topic":"numbers","sources":["fact:S4","ref:ziman2010"],"tags":["gradient","calcium"],"difficulty":2,"url":"/machines/serca#story","cites":[{"source":"fact:S4","machine":"serca","label":"Calcium gradient across the SR membrane: 4,000 times","section":"story"},{"source":"ref:ziman2010","machine":"serca","label":"Ziman 2010","section":"sources","href":"https://doi.org/10.1016/j.bpj.2010.08.032"}]},{"id":"serca-atp-pays","machine":"serca","kind":"qa","prompt":"Why is one ATP enough to pay for SERCA pushing two calcium ions up the SR gradient?","answer":"The push costs about 43 kJ/mol, and one ATP supplies 57–64 kJ/mol in the cell.","explanation":"The 43 kJ/mol is 2 × RT × ln(3,900) at 37 °C; it leaves out any voltage across the SR membrane.","section":"story","topic":"numbers","sources":["fact:S8","ref:bnid100775"],"tags":["energy","atp"],"difficulty":3,"url":"/machines/serca#story","cites":[{"source":"fact:S8","machine":"serca","label":"Energy to push two calcium ions up the gradient: 43 kJ/mol","section":"story"},{"source":"ref:bnid100775","machine":"serca","label":"BNID 100775","section":"sources","href":"https://bionumbers.hms.harvard.edu/bionumber.aspx?id=100775"}]},{"id":"serca-resting-oxygen","machine":"serca","kind":"cloze","prompt":"In resting mouse muscle, SERCA accounts for about {{40–50%}} of the muscle's oxygen use.","answer":"40–50%","explanation":"Measured in isolated mouse muscles at 30 °C. The authors scale this to 12–15% of whole-body resting oxygen use, an estimate.","section":"story","topic":"numbers","sources":["fact:S9","ref:smith2013"],"tags":["energy","metabolism"],"difficulty":2,"url":"/machines/serca#story","cites":[{"source":"fact:S9","machine":"serca","label":"Share of resting muscle oxygen use spent on SERCA: 40–50 %","section":"story"},{"source":"ref:smith2013","machine":"serca","label":"Smith 2013","section":"sources","href":"https://doi.org/10.1371/journal.pone.0068924"}]},{"id":"serca-heart-share","machine":"serca","kind":"cloze","prompt":"In mouse heart muscle cells, SERCA removes about {{90%}} of each beat's calcium.","answer":"90%","explanation":"The sodium-calcium exchanger removes most of the rest (9%), so SERCA sets how fast the heart relaxes.","section":"story","topic":"numbers","sources":["fact:S5","ref:li1998"],"tags":["heart","calcium"],"difficulty":2,"url":"/machines/serca#story","cites":[{"source":"fact:S5","machine":"serca","label":"Share of each beat's calcium that SERCA removes: 90 %","section":"story"},{"source":"ref:li1998","machine":"serca","label":"Li 1998","section":"sources","href":"https://doi.org/10.1152/ajpheart.1998.274.4.h1335"}]},{"id":"serca-airlock-breaks","machine":"serca","kind":"qa","prompt":"SERCA is often compared to an airlock whose two doors never open together. Where does that analogy break?","answer":"An airlock is passive; the energy of ATP drives SERCA's doors.","explanation":"The analogy gets the gating right: because the two gates never open at once, the store cannot leak back.","section":"story","topic":"purpose","sources":["analogy:An airlock"],"tags":["analogy","gating"],"difficulty":2,"url":"/machines/serca#story","cites":[{"source":"analogy:An airlock","machine":"serca","label":"Analogy: An airlock","section":"story"}]},{"id":"serca-myosin-link","machine":"serca","kind":"qa","prompt":"How does SERCA switch myosin off at the end of a contraction?","answer":"It takes away the calcium that switched myosin on.","explanation":"SERCA is the off switch for the calcium signal that myosin, the consumer, responds to.","section":"story","topic":"purpose","sources":["link:myosin"],"tags":["myosin","contraction"],"difficulty":1,"url":"/machines/serca#story","cites":[{"source":"link:myosin","machine":"serca","label":"Link to Myosin","section":"story"}]},{"id":"serca-ratio-method","machine":"serca","kind":"qa","prompt":"How did Yu and Inesi (1995) measure SERCA's calcium-per-ATP ratio?","answer":"By following 45Ca2+ uptake and Ca2+-dependent phosphate release side by side in native rabbit SR vesicles.","explanation":"At the start of pumping, close to two calcium ions went in for each ATP split, and the ratio was never seen above 2.","section":"evidence","topic":"numbers","sources":["evidence:calcium-per-atp-coupling"],"tags":["stoichiometry","method"],"difficulty":3,"url":"/machines/serca#evidence","cites":[{"source":"evidence:calcium-per-atp-coupling","machine":"serca","label":"Ca2+ transported per ATP hydrolysed (Yu X 1995)","section":"evidence","anchor":"ev-calcium-per-atp-coupling"}]},{"id":"serca-ratio-best-case","machine":"serca","kind":"qa","prompt":"Why is two calcium ions per ATP the best case for SERCA rather than a fixed rule?","answer":"The ratio falls as calcium builds up inside the vesicles.","explanation":"With oxalate clamping lumenal calcium, the steady ratio was about 1.5.","section":"evidence","topic":"numbers","sources":["evidence:calcium-per-atp-coupling","evidence:coupling-falls-with-lumenal-calcium"],"tags":["stoichiometry","coupling"],"difficulty":2,"url":"/machines/serca#evidence","cites":[{"source":"evidence:calcium-per-atp-coupling","machine":"serca","label":"Ca2+ transported per ATP hydrolysed (Yu X 1995)","section":"evidence","anchor":"ev-calcium-per-atp-coupling"},{"source":"evidence:coupling-falls-with-lumenal-calcium","machine":"serca","label":"Ca2+/ATP coupling as lumenal Ca2+ rises (Yu X 1995)","section":"evidence","anchor":"ev-coupling-falls-with-lumenal-calcium"}]},{"id":"serca-plateau-not-leak","machine":"serca","kind":"qa","prompt":"After SERCA's calcium uptake into vesicles levelled off, ATP splitting went on. Thapsigargin stopped it, and no calcium leaked out. What did this show?","answer":"The extra ATP went to pumps cycling without moving calcium, not to re-pumping leaked calcium.","explanation":"Lumenal calcium slows the pump itself, so back-inhibition, not leak, limits the gradient SERCA can build.","section":"evidence","topic":"numbers","sources":["evidence:coupling-falls-with-lumenal-calcium","evidence:lumenal-calcium-level"],"tags":["coupling","gradient"],"difficulty":3,"url":"/machines/serca#evidence","cites":[{"source":"evidence:coupling-falls-with-lumenal-calcium","machine":"serca","label":"Ca2+/ATP coupling as lumenal Ca2+ rises (Yu X 1995)","section":"evidence","anchor":"ev-coupling-falls-with-lumenal-calcium"},{"source":"evidence:lumenal-calcium-level","machine":"serca","label":"Lumenal Ca2+ concentration reached by the pump (Yu X 1993)","section":"evidence","anchor":"ev-lumenal-calcium-level"}]},{"id":"serca-proton-ratio","machine":"serca","kind":"cloze","prompt":"In reconstituted proteoliposomes, SERCA moved {{1}} H+ out of the lumen for each Ca2+ moved in.","answer":"1","explanation":"Yu and colleagues (1993) read calcium with murexide and lumenal pH with trapped pyranine; the two traces ran in parallel for 10 minutes. Earlier studies gave 1.0–1.5.","section":"evidence","topic":"numbers","sources":["evidence:proton-countertransport","stat:Proton counter-transport","ref:yu1993"],"tags":["protons","counter-transport"],"difficulty":2,"url":"/machines/serca#evidence","cites":[{"source":"evidence:proton-countertransport","machine":"serca","label":"H+ moved out per Ca2+ moved in (Yu X 1993)","section":"evidence","anchor":"ev-proton-countertransport"},{"source":"stat:Proton counter-transport","machine":"serca","label":"Key number: Proton counter-transport","section":"summary"},{"source":"ref:yu1993","machine":"serca","label":"Yu et al.","section":"sources","anchor":"ref-yu1993","href":"https://doi.org/10.1016/s0006-3495(93)81489-9"}]},{"id":"serca-pln-max-rate","machine":"serca","kind":"qa","prompt":"When purified phospholamban was added to SERCA, how did it change the pump's ATP splitting?","answer":"It shifted the calcium curve to higher calcium (KCa 0.26 to 0.62 µM) without lowering the maximal rate.","explanation":"Phospholamban is a brake on calcium affinity, not on top speed; the superinhibitory mutant PLB4 shifted KCa further, to 1.38 µM.","section":"evidence","topic":"numbers","sources":["evidence:phospholamban-calcium-affinity","ref:akin2013"],"tags":["phospholamban","regulation"],"difficulty":3,"url":"/machines/serca#evidence","cites":[{"source":"evidence:phospholamban-calcium-affinity","machine":"serca","label":"Apparent Ca2+ affinity of the pump (KCa for half-maximal ATPase) with and without phospholamban (Akin BL 2013)","section":"evidence","anchor":"ev-phospholamban-calcium-affinity"},{"source":"ref:akin2013","machine":"serca","label":"Akin et al.","section":"sources","anchor":"ref-akin2013","href":"https://doi.org/10.1074/jbc.M113.501585"}]},{"id":"serca-thapsigargin-tool","machine":"serca","kind":"qa","prompt":"Why did thapsigargin become a standard tool for blocking SERCA?","answer":"It inhibits SERCA's calcium uptake at sub-nanomolar doses but leaves calcium release channels and the plasma-membrane calcium pump unaffected.","explanation":"Free thapsigargin at 0.1 nM already inhibits uptake in SR vesicles; it binds the calcium-free pump and blocks calcium binding.","section":"evidence","topic":"numbers","sources":["evidence:thapsigargin-subnanomolar","stat:Thapsigargin inhibition","ref:sagara1991"],"tags":["thapsigargin","inhibitor"],"difficulty":2,"url":"/machines/serca#evidence","cites":[{"source":"evidence:thapsigargin-subnanomolar","machine":"serca","label":"Lowest free thapsigargin concentration that inhibits Ca2+ uptake (Sagara Y 1991)","section":"evidence","anchor":"ev-thapsigargin-subnanomolar"},{"source":"stat:Thapsigargin inhibition","machine":"serca","label":"Key number: Thapsigargin inhibition","section":"summary"},{"source":"ref:sagara1991","machine":"serca","label":"Sagara and Inesi, J Biol Chem 1991","section":"sources","anchor":"ref-sagara1991","href":"https://doi.org/10.1016/s0021-9258(18)92726-2"}]},{"id":"serca-monomer-method","machine":"serca","kind":"qa","prompt":"How did Heegaard and colleagues show that a single SERCA chain can pump calcium on its own?","answer":"In lipid-rich vesicles that still pumped, freeze-fracture EM counted one membrane particle per chain, and cross-linking found no pump pairs.","explanation":"So one polypeptide is the working unit; regulators such as phospholamban bind from the membrane.","section":"evidence","topic":"parts","sources":["evidence:monomer-transports-calcium","stat:Chains in the working unit","ref:heegaard1990"],"tags":["monomer","method"],"difficulty":3,"url":"/machines/serca#evidence","cites":[{"source":"evidence:monomer-transports-calcium","machine":"serca","label":"Oligomeric state of a pump that transports Ca2+ (Heegaard CW 1990)","section":"evidence","anchor":"ev-monomer-transports-calcium"},{"source":"stat:Chains in the working unit","machine":"serca","label":"Key number: Chains in the working unit","section":"summary"},{"source":"ref:heegaard1990","machine":"serca","label":"Heegaard et al.","section":"sources","anchor":"ref-heegaard1990","href":"https://doi.org/10.1016/S0021-9258(19)38502-3"}]},{"id":"hemoglobin-job","machine":"hemoglobin","kind":"qa","prompt":"What does hemoglobin carry, and from where to where?","answer":"Oxygen, from the lungs to the tissues.","explanation":"Hemoglobin loads oxygen in the lungs and unloads it where tissues need it, because it switches shape as a whole.","section":"summary","topic":"purpose","sources":["machine:summary","machine:tagline"],"tags":["oxygen","transport"],"difficulty":1,"url":"/machines/hemoglobin#summary","cites":[{"source":"machine:summary","machine":"hemoglobin","label":"Summary","section":"summary"},{"source":"machine:tagline","machine":"hemoglobin","label":"Summary","section":"summary"}]},{"id":"hemoglobin-no-fuel","machine":"hemoglobin","kind":"qa","prompt":"Hemoglobin spends no chemical fuel. What does the work of loading and unloading oxygen instead?","answer":"The oxygen gradient and the allosteric switch.","explanation":"SERCA also switches between two states, but it spends ATP to do so; hemoglobin spends nothing.","section":"summary","topic":"purpose","sources":["machine:energy","link:serca"],"tags":["energy","allostery"],"difficulty":2,"url":"/machines/hemoglobin#summary","cites":[{"source":"machine:energy","machine":"hemoglobin","label":"Summary","section":"summary"},{"source":"link:serca","machine":"hemoglobin","label":"Link to SERCA calcium pump","section":"story"}]},{"id":"hemoglobin-two-dimers","machine":"hemoglobin","kind":"cloze","prompt":"Human adult hemoglobin has two alpha and two beta chains, arranged as a pair of {{alpha-beta dimers}}.","answer":"alpha-beta dimers","explanation":"Each dimer changes little inside itself, so the T-to-R switch is one dimer moving against the other.","section":"summary","topic":"parts","sources":["stat:Chains in the working unit","evidence:human-hb-tetramer-two-dimers","ref:fermi1984"],"tags":["structure","subunits"],"difficulty":1,"url":"/machines/hemoglobin#summary","cites":[{"source":"stat:Chains in the working unit","machine":"hemoglobin","label":"Key number: Chains in the working unit","section":"summary"},{"source":"evidence:human-hb-tetramer-two-dimers","machine":"hemoglobin","label":"Subunit arrangement of the human hemoglobin tetramer (Takahashi K 2024)","section":"evidence","anchor":"ev-human-hb-tetramer-two-dimers"},{"source":"ref:fermi1984","machine":"hemoglobin","label":"Fermi et al.","section":"sources","anchor":"ref-fermi1984","href":"https://doi.org/10.1016/0022-2836(84)90472-8"}]},{"id":"hemoglobin-sigmoid-why","machine":"hemoglobin","kind":"qa","prompt":"Why is hemoglobin's oxygen binding curve sigmoid?","answer":"Its four sites are not independent: the tetramer switches from a low-affinity T state to a high-affinity R state.","explanation":"Early oxygens bind weakly to T; once the tetramer snaps to R, the remaining hemes bind with much higher affinity.","section":"summary","topic":"purpose","sources":["machine:summary","mechanism:The last oxygens bind","ref:monod1965"],"tags":["cooperativity","allostery"],"difficulty":2,"url":"/machines/hemoglobin#summary","cites":[{"source":"machine:summary","machine":"hemoglobin","label":"Summary","section":"summary"},{"source":"mechanism:The last oxygens bind","machine":"hemoglobin","label":"Step: The last oxygens bind","section":"mechanism"},{"source":"ref:monod1965","machine":"hemoglobin","label":"Monod et al.","section":"sources","anchor":"ref-monod1965","href":"https://doi.org/10.1016/S0022-2836(65)80285-6"}]},{"id":"hemoglobin-hill-value","machine":"hemoglobin","kind":"cloze","prompt":"Unmodified, stroma-free human hemoglobin at pH 7.40 and 37 °C has a Hill coefficient of {{2.8}}.","answer":"2.8","explanation":"A Hill coefficient of 1 would mean each site binds on its own, so 2.8 means the first oxygens to bind make the others bind more tightly. Phosphate buffer at 29 °C gives values near 3.1.","section":"summary","topic":"numbers","sources":["stat:Hill coefficient","evidence:hill-coefficient-unmodified-hb","ref:vanderplas1988"],"tags":["cooperativity","hill"],"difficulty":2,"url":"/machines/hemoglobin#summary","cites":[{"source":"stat:Hill coefficient","machine":"hemoglobin","label":"Key number: Hill coefficient","section":"summary"},{"source":"evidence:hill-coefficient-unmodified-hb","machine":"hemoglobin","label":"Hill coefficient of unmodified human hemoglobin in solution (van der Plas J 1988)","section":"evidence","anchor":"ev-hill-coefficient-unmodified-hb"},{"source":"ref:vanderplas1988","machine":"hemoglobin","label":"van der Plas et al.","section":"sources","anchor":"ref-vanderplas1988","href":"https://doi.org/10.1046/j.1537-2995.1988.28689059024.x"}]},{"id":"hemoglobin-fetal-gamma","machine":"hemoglobin","kind":"qa","prompt":"Why does fetal hemoglobin hold oxygen more tightly than adult hemoglobin?","answer":"Its gamma chains, which replace beta, bind 2,3-BPG only weakly.","explanation":"2,3-BPG lowers oxygen affinity, so weak binding moves the fetal curve left and fetal blood can take oxygen from maternal blood.","section":"summary","topic":"parts","sources":["component:gamma","species:Human fetal hemoglobin","ref:sankaran2013"],"tags":["fetal","2,3-bpg"],"difficulty":2,"url":"/machines/hemoglobin#summary","cites":[{"source":"component:gamma","machine":"hemoglobin","label":"Part: gamma","section":"summary"},{"source":"species:Human fetal hemoglobin","machine":"hemoglobin","label":"Human fetal hemoglobin","section":"summary"},{"source":"ref:sankaran2013","machine":"hemoglobin","label":"Sankaran and Orkin, Cold Spring Harb Perspect Med 2013","section":"sources","anchor":"ref-sankaran2013","href":"https://doi.org/10.1101/cshperspect.a011643"}]},{"id":"hemoglobin-global-allostery","machine":"hemoglobin","kind":"qa","prompt":"In the global allostery model of hemoglobin, what carries much of the affinity change and most of the Bohr effect?","answer":"Tertiary changes inside both the T and the R state.","explanation":"Broader oxygen-binding measurements support this model (status: demonstrated). The two-state model stays useful as a compact fit, not as the final mechanism.","section":"summary","topic":"debate","sources":["frontier:Beyond two states","ref:yonetani2003"],"tags":["allostery","models"],"difficulty":3,"url":"/machines/hemoglobin#summary","cites":[{"source":"frontier:Beyond two states","machine":"hemoglobin","label":"Open question: Beyond two states","section":"summary"},{"source":"ref:yonetani2003","machine":"hemoglobin","label":"Yonetani and Tsuneshige, C R Biol 2003","section":"sources","anchor":"ref-yonetani2003","href":"https://doi.org/10.1016/s1631-0691(03)00150-1"}]},{"id":"hemoglobin-designed-switch","machine":"hemoglobin","kind":"qa","prompt":"Hemoglobin shows the minimum parts for an allosteric switch. What are the three parts?","answer":"A rigid dimer, one interface with exactly two good arrangements, and a ligand site that senses which arrangement it occupies.","explanation":"Designed allosteric switches aim at the same set of parts; this is a proposal, not yet a result.","section":"summary","topic":"debate","sources":["frontier:Designed allosteric switches","ref:baldwin1979"],"tags":["design","allostery"],"difficulty":3,"url":"/machines/hemoglobin#summary","cites":[{"source":"frontier:Designed allosteric switches","machine":"hemoglobin","label":"Open question: Designed allosteric switches","section":"summary"},{"source":"ref:baldwin1979","machine":"hemoglobin","label":"Baldwin and Chothia, J Mol Biol 1979","section":"sources","anchor":"ref-baldwin1979","href":"https://doi.org/10.1016/0022-2836(79)90277-8"}]},{"id":"hemoglobin-iron-into-plane","machine":"hemoglobin","kind":"cloze","prompt":"When the first oxygen binds a hemoglobin heme, the iron moves {{into the heme plane}}.","answer":"into the heme plane","explanation":"In the T state without oxygen, the iron sits slightly out of the heme plane. Its move is the local trigger of the switch.","section":"mechanism","topic":"cycle","sources":["mechanism:The first oxygen binds","mechanism:The T state waits","ref:perutz1970"],"tags":["heme","iron"],"difficulty":1,"url":"/machines/hemoglobin#mechanism","cites":[{"source":"mechanism:The first oxygen binds","machine":"hemoglobin","label":"Step: The first oxygen binds","section":"mechanism"},{"source":"mechanism:The T state waits","machine":"hemoglobin","label":"Step: The T state waits","section":"mechanism"},{"source":"ref:perutz1970","machine":"hemoglobin","label":"Perutz, Nature 1970","section":"sources","anchor":"ref-perutz1970","href":"https://doi.org/10.1038/228726a0"}]},{"id":"hemoglobin-proximal-histidine","machine":"hemoglobin","kind":"qa","prompt":"In hemoglobin, what links the heme iron to the protein, so that the iron's move pulls on a helix?","answer":"The proximal histidine.","explanation":"The iron binds oxygen on one side and the proximal histidine on the other. As the iron moves into the plane, it drags the histidine and its helix along: a tertiary change inside one subunit.","section":"mechanism","topic":"parts","sources":["mechanism:The first oxygen binds","component:heme","ref:perutz1970"],"tags":["heme","histidine"],"difficulty":2,"url":"/machines/hemoglobin#mechanism","cites":[{"source":"mechanism:The first oxygen binds","machine":"hemoglobin","label":"Step: The first oxygen binds","section":"mechanism"},{"source":"component:heme","machine":"hemoglobin","label":"Part: heme","section":"summary"},{"source":"ref:perutz1970","machine":"hemoglobin","label":"Perutz, Nature 1970","section":"sources","anchor":"ref-perutz1970","href":"https://doi.org/10.1038/228726a0"}]},{"id":"hemoglobin-switch-contact","machine":"hemoglobin","kind":"cloze","prompt":"In hemoglobin, the {{alpha1-beta2}} contact is the switch point between the T and R quaternary states.","answer":"alpha1-beta2","explanation":"The helix shift from a bound heme travels to this contact, so local binding becomes a global signal.","section":"mechanism","topic":"cycle","sources":["mechanism:The change reaches the dimer interface","ref:baldwin1979"],"tags":["interface","allostery"],"difficulty":2,"url":"/machines/hemoglobin#mechanism","cites":[{"source":"mechanism:The change reaches the dimer interface","machine":"hemoglobin","label":"Step: The change reaches the dimer interface","section":"mechanism"},{"source":"ref:baldwin1979","machine":"hemoglobin","label":"Baldwin and Chothia, J Mol Biol 1979","section":"sources","anchor":"ref-baldwin1979","href":"https://doi.org/10.1016/0022-2836(79)90277-8"}]},{"id":"hemoglobin-quaternary-turn","machine":"hemoglobin","kind":"cloze","prompt":"When hemoglobin snaps from T to R, one alpha-beta dimer rotates by about {{14 degrees}} against the other.","answer":"14 degrees","explanation":"The site measured 14.2 degrees from 2HHB to 2DN3; a published analysis gives 15.0 degrees. The turn comes with a small screw translation of about 1.4 A.","section":"mechanism","topic":"numbers","sources":["mechanism:The tetramer snaps to R","stat:Quaternary turn, T to R","ref:baldwin1979","ref:takahashi2024"],"tags":["quaternary","rotation"],"difficulty":2,"url":"/machines/hemoglobin#mechanism","cites":[{"source":"mechanism:The tetramer snaps to R","machine":"hemoglobin","label":"Step: The tetramer snaps to R","section":"mechanism"},{"source":"stat:Quaternary turn, T to R","machine":"hemoglobin","label":"Key number: Quaternary turn, T to R","section":"summary"},{"source":"ref:baldwin1979","machine":"hemoglobin","label":"Baldwin and Chothia, J Mol Biol 1979","section":"sources","anchor":"ref-baldwin1979","href":"https://doi.org/10.1016/0022-2836(79)90277-8"},{"source":"ref:takahashi2024","machine":"hemoglobin","label":"Takahashi et al.","section":"sources","anchor":"ref-takahashi2024","href":"https://doi.org/10.1038/s41467-024-49947-x"}]},{"id":"hemoglobin-tissue-effectors","machine":"hemoglobin","kind":"qa","prompt":"Which three small molecules in working tissue push hemoglobin back toward the T state?","answer":"Protons, carbon dioxide and 2,3-BPG.","explanation":"They bind the T state better than the R state, so the equilibrium shifts back to T and oxygen comes off where it is needed.","section":"mechanism","topic":"cycle","sources":["mechanism:Tissues push the switch back","ref:benesch1967"],"tags":["effectors","bohr"],"difficulty":2,"url":"/machines/hemoglobin#mechanism","cites":[{"source":"mechanism:Tissues push the switch back","machine":"hemoglobin","label":"Step: Tissues push the switch back","section":"mechanism"},{"source":"ref:benesch1967","machine":"hemoglobin","label":"Benesch and Benesch, Biochem Biophys Res Commun 1967","section":"sources","anchor":"ref-benesch1967","href":"https://doi.org/10.1016/0006-291x(67)90228-8"}]},{"id":"hemoglobin-bpg-favours-t","machine":"hemoglobin","kind":"qa","prompt":"Why does 2,3-BPG favour the T state of hemoglobin?","answer":"It binds the central cavity between the beta chains, which is wide enough only in the T state.","explanation":"The central cavity narrows when the tetramer snaps to R. By binding T better than R, 2,3-BPG lowers oxygen affinity.","section":"mechanism","topic":"cycle","sources":["evidence:dpg-binding-site-low-salt","mechanism:The tetramer snaps to R","ref:richard1993"],"tags":["2,3-bpg","cavity"],"difficulty":3,"url":"/machines/hemoglobin#mechanism","cites":[{"source":"evidence:dpg-binding-site-low-salt","machine":"hemoglobin","label":"Contacts made by 2,3-diphosphoglycerate in the central cavity of deoxyhemoglobin (Richard V 1993)","section":"evidence","anchor":"ev-dpg-binding-site-low-salt"},{"source":"mechanism:The tetramer snaps to R","machine":"hemoglobin","label":"Step: The tetramer snaps to R","section":"mechanism"},{"source":"ref:richard1993","machine":"hemoglobin","label":"Richard et al.","section":"sources","anchor":"ref-richard1993","href":"https://doi.org/10.1006/jmbi.1993.1505"}]},{"id":"hemoglobin-myoglobin-no-switch","machine":"hemoglobin","kind":"qa","prompt":"Myoglobin has the same globin fold as hemoglobin. Why does myoglobin show no cooperativity?","answer":"It has one chain and one heme, so it has no dimer interface where a tertiary change could become a quaternary one.","explanation":"Myoglobin keeps the ancestral job of storing and buffering oxygen in muscle; it is the control case for cooperativity.","section":"mechanism","topic":"parts","sources":["evolution:Cooperativity after the monomer","species:Myoglobin","ref:hardison2012"],"tags":["myoglobin","evolution"],"difficulty":3,"url":"/machines/hemoglobin#mechanism","cites":[{"source":"evolution:Cooperativity after the monomer","machine":"hemoglobin","label":"Cooperativity after the monomer","section":"summary"},{"source":"species:Myoglobin","machine":"hemoglobin","label":"Myoglobin","section":"summary"},{"source":"ref:hardison2012","machine":"hemoglobin","label":"Hardison, Cold Spring Harb Perspect Med 2012","section":"sources","anchor":"ref-hardison2012","href":"https://doi.org/10.1101/cshperspect.a011627"}]},{"id":"hemoglobin-per-red-cell","machine":"hemoglobin","kind":"cloze","prompt":"One human red blood cell holds about {{270 million}} hemoglobin molecules.","answer":"270 million","explanation":"At 4 oxygen per hemoglobin, a full red cell carries about 1.1 x 10^9 oxygen molecules.","section":"story","topic":"numbers","sources":["fact:N8","stop:rbc","fact:N18","ref:bnid102740"],"tags":["red-cell","scale"],"difficulty":2,"url":"/machines/hemoglobin#story","cites":[{"source":"fact:N8","machine":"hemoglobin","label":"Hemoglobin molecules in one red cell: 2.7 × 10^8","section":"story"},{"source":"stop:rbc","machine":"hemoglobin","label":"Big picture: Red blood cell","section":"story"},{"source":"fact:N18","machine":"hemoglobin","label":"Oxygen molecules in one full red cell: 1.1 × 10^9 O2","section":"story"},{"source":"ref:bnid102740","machine":"hemoglobin","label":"BNID 102740","section":"sources","href":"https://bionumbers.hms.harvard.edu/bionumber.aspx?id=102740"}]},{"id":"hemoglobin-rbc-no-burn","machine":"hemoglobin","kind":"qa","prompt":"Why does a red blood cell not burn the oxygen its hemoglobin carries?","answer":"It has no mitochondria.","explanation":"A mature red cell has thrown out its nucleus and mitochondria; it is little more than a bag of hemoglobin.","section":"story","topic":"purpose","sources":["fact:N9","stop:rbc","ref:openstax-ap2e-18-3"],"tags":["red-cell"],"difficulty":1,"url":"/machines/hemoglobin#story","cites":[{"source":"fact:N9","machine":"hemoglobin","label":"Nucleus and mitochondria in a mature red cell: None","section":"story"},{"source":"stop:rbc","machine":"hemoglobin","label":"Big picture: Red blood cell","section":"story"},{"source":"ref:openstax-ap2e-18-3","machine":"hemoglobin","label":"OpenStax A&P 18.3","section":"sources","href":"https://openstax.org/books/anatomy-and-physiology-2e"}]},{"id":"hemoglobin-share-of-blood-oxygen","machine":"hemoglobin","kind":"cloze","prompt":"Hemoglobin carries about {{98.5%}} of the oxygen in blood; the rest is dissolved.","answer":"98.5%","explanation":"Water holds only a little oxygen, so blood packs almost all of it into hemoglobin. This is a textbook value.","section":"story","topic":"purpose","sources":["fact:N2","stop:body","ref:openstax-bio2e-39-4"],"tags":["oxygen","blood"],"difficulty":2,"url":"/machines/hemoglobin#story","cites":[{"source":"fact:N2","machine":"hemoglobin","label":"Share of blood oxygen carried by hemoglobin: 98.5 %","section":"story"},{"source":"stop:body","machine":"hemoglobin","label":"Big picture: You, breathing","section":"story"},{"source":"ref:openstax-bio2e-39-4","machine":"hemoglobin","label":"OpenStax Biology 2e","section":"sources","href":"https://openstax.org/books/biology-2e"}]},{"id":"hemoglobin-rest-extraction","machine":"hemoglobin","kind":"qa","prompt":"At rest, roughly what share of the oxygen delivered by hemoglobin do the body's tissues take?","answer":"About a quarter.","explanation":"Blood returns to the lungs about 74% saturated (a calculation, not a measurement), which leaves a reserve for when you run.","section":"story","topic":"numbers","sources":["fact:N16","stop:muscle"],"tags":["reserve","exercise"],"difficulty":2,"url":"/machines/hemoglobin#story","cites":[{"source":"fact:N16","machine":"hemoglobin","label":"Oxygen saturation of blood that returns from the body at rest: 74 %","section":"story"},{"source":"stop:muscle","machine":"hemoglobin","label":"Big picture: Working muscle","section":"story"}]},{"id":"hemoglobin-van-analogy-breaks","machine":"hemoglobin","kind":"qa","prompt":"Hemoglobin is like a delivery van with four seats that loads and unloads as a team. Where does that analogy break?","answer":"The seats do not fill one by one; the whole molecule flips between T and R, and that shape sets the grip of all four seats at once.","explanation":"The analogy gets the teamwork right: once some seats fill, the rest fill more easily. There is also no driver and no fuel.","section":"story","topic":"purpose","sources":["analogy:A delivery van with four seats that loads and unloads as a team"],"tags":["analogy","allostery"],"difficulty":2,"url":"/machines/hemoglobin#story","cites":[{"source":"analogy:A delivery van with four seats that loads and unloads as a team","machine":"hemoglobin","label":"Analogy: A delivery van with four seats that loads and unloads as a team","section":"story"}]},{"id":"hemoglobin-cas9-fetal","machine":"hemoglobin","kind":"qa","prompt":"How does an approved Cas9 therapy use hemoglobin to treat sickle cell disease?","answer":"It edits a person's blood stem cells so their red cells make fetal hemoglobin.","explanation":"Turning gamma back on treats sickle cell disease and beta-thalassaemia while leaving the hemoglobin protein untouched.","section":"story","topic":"purpose","sources":["link:cas9","frontier:Re-induced fetal hemoglobin","ref:frangoul2021","ref:sankaran2013"],"tags":["cas9","sickle","fetal"],"difficulty":2,"url":"/machines/hemoglobin#story","cites":[{"source":"link:cas9","machine":"hemoglobin","label":"Link to Cas9","section":"story"},{"source":"frontier:Re-induced fetal hemoglobin","machine":"hemoglobin","label":"Open question: Re-induced fetal hemoglobin","section":"summary"},{"source":"ref:frangoul2021","machine":"hemoglobin","label":"Frangoul 2021","section":"sources","href":"https://doi.org/10.1056/NEJMoa2031054"},{"source":"ref:sankaran2013","machine":"hemoglobin","label":"Sankaran and Orkin, Cold Spring Harb Perspect Med 2013","section":"sources","anchor":"ref-sankaran2013","href":"https://doi.org/10.1101/cshperspect.a011643"}]},{"id":"hemoglobin-p50-standard","machine":"hemoglobin","kind":"cloze","prompt":"Human blood under standard conditions (pH 7.4, pCO2 40 mmHg, 37 °C) has a P50 of {{26.6 mmHg}}.","answer":"26.6 mmHg","explanation":"P50 is the oxygen pressure at which half the hemoglobin carries oxygen; a higher P50 means blood gives up oxygen more easily. Clerbaux and colleagues measured it (26.6 +/- 1.2 mmHg) from whole dissociation curves of fresh blood.","section":"evidence","topic":"numbers","sources":["evidence:p50-human-standard","stat:P50, standard conditions","ref:clerbaux1993"],"tags":["p50","affinity"],"difficulty":2,"url":"/machines/hemoglobin#evidence","cites":[{"source":"evidence:p50-human-standard","machine":"hemoglobin","label":"Oxygen pressure at half saturation of human blood under standard conditions (standard P50) (Clerbaux T 1993)","section":"evidence","anchor":"ev-p50-human-standard"},{"source":"stat:P50, standard conditions","machine":"hemoglobin","label":"Key number: P50, standard conditions","section":"summary"},{"source":"ref:clerbaux1993","machine":"hemoglobin","label":"Clerbaux et al.","section":"sources","anchor":"ref-clerbaux1993","href":"https://doi.org/10.1016/0300-9629(93)90382-e"}]},{"id":"hemoglobin-hill-steepness","machine":"hemoglobin","kind":"qa","prompt":"With a Hill coefficient of 2.8, by what factor must oxygen pressure rise to take hemoglobin from 10% to 90% saturation?","answer":"About 4.8-fold (against 81-fold with no cooperativity).","explanation":"Cooperativity makes the curve steep: the first oxygens to bind make the others bind more tightly. Van der Plas and colleagues measured n = 2.8 on purified human hemoglobin.","section":"evidence","topic":"numbers","sources":["evidence:hill-coefficient-unmodified-hb","ref:vanderplas1988"],"tags":["cooperativity","hill"],"difficulty":3,"url":"/machines/hemoglobin#evidence","cites":[{"source":"evidence:hill-coefficient-unmodified-hb","machine":"hemoglobin","label":"Hill coefficient of unmodified human hemoglobin in solution (van der Plas J 1988)","section":"evidence","anchor":"ev-hill-coefficient-unmodified-hb"},{"source":"ref:vanderplas1988","machine":"hemoglobin","label":"van der Plas et al.","section":"sources","anchor":"ref-vanderplas1988","href":"https://doi.org/10.1046/j.1537-2995.1988.28689059024.x"}]},{"id":"hemoglobin-bohr-meaning","machine":"hemoglobin","kind":"qa","prompt":"Human hemoglobin has a Bohr coefficient of -0.29. What does a pH drop of 0.1 unit do to its P50?","answer":"Raises it by about 7 percent.","explanation":"Van der Plas and colleagues took the slope of log P50 against pH over pH 7.1 to 7.7. Acid from working muscle therefore makes hemoglobin release more oxygen.","section":"evidence","topic":"numbers","sources":["evidence:bohr-coefficient-unmodified-hb","stat:Bohr coefficient","ref:vanderplas1988"],"tags":["bohr","ph"],"difficulty":3,"url":"/machines/hemoglobin#evidence","cites":[{"source":"evidence:bohr-coefficient-unmodified-hb","machine":"hemoglobin","label":"Proton Bohr factor of unmodified human hemoglobin (van der Plas J 1988)","section":"evidence","anchor":"ev-bohr-coefficient-unmodified-hb"},{"source":"stat:Bohr coefficient","machine":"hemoglobin","label":"Key number: Bohr coefficient","section":"summary"},{"source":"ref:vanderplas1988","machine":"hemoglobin","label":"van der Plas et al.","section":"sources","anchor":"ref-vanderplas1988","href":"https://doi.org/10.1046/j.1537-2995.1988.28689059024.x"}]},{"id":"hemoglobin-iron-out-of-plane","machine":"hemoglobin","kind":"qa","prompt":"In crystals of human deoxyhemoglobin, how far does the heme iron sit from the porphyrin plane?","answer":"About 0.4 A: 0.40 A in the alpha hemes, 0.36 A in the beta hemes.","explanation":"Fermi and colleagues measured this by X-ray crystallography at 1.74 A. In oxyhemoglobin the iron sits almost in the plane; that shift is the trigger the Perutz mechanism builds on.","section":"evidence","topic":"numbers","sources":["evidence:iron-out-of-plane-deoxy","fact:N13","ref:fermi1984"],"tags":["heme","iron","x-ray"],"difficulty":3,"url":"/machines/hemoglobin#evidence","cites":[{"source":"evidence:iron-out-of-plane-deoxy","machine":"hemoglobin","label":"Displacement of the heme iron from the porphyrin nitrogen plane in deoxyhemoglobin (Fermi G 1984)","section":"evidence","anchor":"ev-iron-out-of-plane-deoxy"},{"source":"fact:N13","machine":"hemoglobin","label":"Distance of the iron from the heme plane without oxygen: 0.36–0.40 Å","section":"story"},{"source":"ref:fermi1984","machine":"hemoglobin","label":"Fermi et al.","section":"sources","anchor":"ref-fermi1984","href":"https://doi.org/10.1016/0022-2836(84)90472-8"}]},{"id":"hemoglobin-t-state-oxy-crystal","machine":"hemoglobin","kind":"qa","prompt":"When T-state hemoglobin crystals were oxygenated at all four hemes, what moved toward R while the tetramer stayed T?","answer":"The heme pockets and the alpha1-beta2 interface.","explanation":"The crystal lattice held the tetramer in T, separating the tertiary change from the quaternary switch. The hemes affect each other even inside the T state.","section":"evidence","topic":"cycle","sources":["evidence:four-hemes-bind-oxygen","ref:paoli1996"],"tags":["t-state","x-ray"],"difficulty":3,"url":"/machines/hemoglobin#evidence","cites":[{"source":"evidence:four-hemes-bind-oxygen","machine":"hemoglobin","label":"Number of heme sites that bind oxygen in one tetramer, shown with the T quaternary structure kept (Paoli M 1996)","section":"evidence","anchor":"ev-four-hemes-bind-oxygen"},{"source":"ref:paoli1996","machine":"hemoglobin","label":"Paoli et al.","section":"sources","anchor":"ref-paoli1996","href":"https://doi.org/10.1006/jmbi.1996.0124"}]},{"id":"hemoglobin-effectors-in-r","machine":"hemoglobin","kind":"qa","prompt":"In R-state crystals of horse hemoglobin, what did the effector bezafibrate do to oxygen affinity?","answer":"Lowered it about threefold (P50 from 0.32 to 0.91 torr), with binding still non-cooperative.","explanation":"The lattice blocks the switch to T, so effectors can weaken the R state itself, not only shift the T-R balance as the two-state model assumes.","section":"evidence","topic":"debate","sources":["evidence:effectors-act-inside-the-r-state","frontier:Beyond two states","ref:shibayama2025"],"tags":["effectors","r-state","models"],"difficulty":3,"url":"/machines/hemoglobin#evidence","cites":[{"source":"evidence:effectors-act-inside-the-r-state","machine":"hemoglobin","label":"Change in R-state oxygen affinity caused by allosteric effectors, with the quaternary structure held fixed (Shibayama N 2025)","section":"evidence","anchor":"ev-effectors-act-inside-the-r-state"},{"source":"frontier:Beyond two states","machine":"hemoglobin","label":"Open question: Beyond two states","section":"summary"},{"source":"ref:shibayama2025","machine":"hemoglobin","label":"Shibayama, Protein Sci 2025","section":"sources","anchor":"ref-shibayama2025","href":"https://doi.org/10.1002/pro.5232"}]},{"id":"hemoglobin-cryoem-r2","machine":"hemoglobin","kind":"cloze","prompt":"Cryo-EM puts liganded human hemoglobin, free of crystal contacts, closest to the {{R2}} quaternary arrangement.","answer":"R2","explanation":"Its dimers turn 22.1 degrees against deoxy hemoglobin, against 15.0 degrees for the R crystal form, so the single R state of the two-state picture is a simplification.","section":"evidence","topic":"debate","sources":["evidence:liganded-human-hb-closer-to-r2","frontier:Beyond two states","ref:takahashi2024"],"tags":["cryo-em","r2","models"],"difficulty":3,"url":"/machines/hemoglobin#evidence","cites":[{"source":"evidence:liganded-human-hb-closer-to-r2","machine":"hemoglobin","label":"Quaternary position of liganded human hemoglobin measured by cryo-EM in solution-like conditions (Takahashi K 2024)","section":"evidence","anchor":"ev-liganded-human-hb-closer-to-r2"},{"source":"frontier:Beyond two states","machine":"hemoglobin","label":"Open question: Beyond two states","section":"summary"},{"source":"ref:takahashi2024","machine":"hemoglobin","label":"Takahashi et al.","section":"sources","anchor":"ref-takahashi2024","href":"https://doi.org/10.1038/s41467-024-49947-x"}]},{"id":"cas9-job","machine":"cas9","kind":"qa","prompt":"What does Cas9 do for the bacterium that carries it?","answer":"It destroys invading DNA.","explanation":"Cas9 is a bacterial immune protein. It cuts the DNA of an invader, so the cell survives the attack.","section":"summary","topic":"purpose","sources":["machine:summary","machine:tagline"],"tags":["immunity","dna"],"difficulty":1,"url":"/machines/cas9#summary","cites":[{"source":"machine:summary","machine":"cas9","label":"Summary","section":"summary"},{"source":"machine:tagline","machine":"cas9","label":"Summary","section":"summary"}]},{"id":"cas9-address-in-rna","machine":"cas9","kind":"qa","prompt":"In Cas9, what part of the machine holds the address of the target?","answer":"The guide RNA, not the protein.","explanation":"Because the address sits in the RNA, one enzyme reaches any sequence: a new guide gives a new target.","section":"summary","topic":"purpose","sources":["machine:summary"],"tags":["guide-rna","targeting"],"difficulty":1,"url":"/machines/cas9#summary","cites":[{"source":"machine:summary","machine":"cas9","label":"Summary","section":"summary"}]},{"id":"cas9-energy","machine":"cas9","kind":"cloze","prompt":"Cas9 spends no ATP on targeting; cleavage needs {{Mg2+}} and the free energy of RNA-DNA base pairing.","answer":"Mg2+","explanation":"Nothing in the search or the cut is paid for with ATP. The metal serves the two nuclease sites.","section":"summary","topic":"purpose","sources":["machine:energy"],"tags":["energy","magnesium"],"difficulty":2,"url":"/machines/cas9#summary","cites":[{"source":"machine:energy","machine":"cas9","label":"Summary","section":"summary"}]},{"id":"cas9-hnh-strand","machine":"cas9","kind":"qa","prompt":"Which DNA strand does the HNH domain of Cas9 cut?","answer":"The target strand, the one that base pairs with the guide.","explanation":"HNH, residues 770-921, carries His840 as its proton acceptor. Its position, not its chemistry, decides whether cleavage happens.","section":"summary","topic":"parts","sources":["component:HNH","stat:Nuclease domains","ref:jinek2012"],"tags":["hnh","domains"],"difficulty":1,"url":"/machines/cas9#summary","cites":[{"source":"component:HNH","machine":"cas9","label":"Part: HNH","section":"summary"},{"source":"stat:Nuclease domains","machine":"cas9","label":"Key number: Nuclease domains","section":"summary"},{"source":"ref:jinek2012","machine":"cas9","label":"Jinek et al.","section":"sources","anchor":"ref-jinek2012","href":"https://doi.org/10.1126/science.1225829"}]},{"id":"cas9-ruvc-strand","machine":"cas9","kind":"qa","prompt":"Which DNA strand does the RuvC domain of Cas9 cut?","answer":"The non-target strand, the displaced strand that carries the PAM.","explanation":"RuvC is built from three segments of the chain (1-62, 718-765 and 925-1102) and uses Asp10 as its catalytic residue.","section":"summary","topic":"parts","sources":["component:RuvC","component:non-target DNA strand","ref:jinek2012"],"tags":["ruvc","domains"],"difficulty":2,"url":"/machines/cas9#summary","cites":[{"source":"component:RuvC","machine":"cas9","label":"Part: RuvC","section":"summary"},{"source":"component:non-target DNA strand","machine":"cas9","label":"Part: non-target DNA strand","section":"summary"},{"source":"ref:jinek2012","machine":"cas9","label":"Jinek et al.","section":"sources","anchor":"ref-jinek2012","href":"https://doi.org/10.1126/science.1225829"}]},{"id":"cas9-catalytic-residues","machine":"cas9","kind":"cloze","prompt":"The two catalytic residues of SpCas9 are {{D10 and H840}}.","answer":"D10 and H840","explanation":"Asp10 serves the RuvC-like site and His840 is the proton acceptor of the HNH site, so changing both removes all cutting.","section":"summary","topic":"numbers","sources":["stat:Catalytic residues","ref:uniprot_q99zw2"],"tags":["active-site","residues"],"difficulty":2,"url":"/machines/cas9#summary","cites":[{"source":"stat:Catalytic residues","machine":"cas9","label":"Key number: Catalytic residues","section":"summary"},{"source":"ref:uniprot_q99zw2","machine":"cas9","label":"UniProt Q99ZW2 (CAS9_STRP1), Streptococcus pyogenes serotype M1","section":"sources","anchor":"ref-uniprot_q99zw2","href":"https://rest.uniprot.org/uniprotkb/Q99ZW2.json"}]},{"id":"cas9-pam-arginines","machine":"cas9","kind":"qa","prompt":"Which two Cas9 residues read the two guanines of the NGG PAM?","answer":"Arg1333 and Arg1335.","explanation":"The PAM is the protospacer adjacent motif. Reading it limits which sites the enzyme can reach.","section":"summary","topic":"parts","sources":["stat:PAM required","fact:s-pam","ref:anders2014"],"tags":["pam","residues"],"difficulty":2,"url":"/machines/cas9#summary","cites":[{"source":"stat:PAM required","machine":"cas9","label":"Key number: PAM required","section":"summary"},{"source":"fact:s-pam","machine":"cas9","label":"Arginines that read the PAM: 2 arginines","section":"story"},{"source":"ref:anders2014","machine":"cas9","label":"Anders et al.","section":"sources","anchor":"ref-anders2014","href":"https://doi.org/10.1038/nature13579"}]},{"id":"cas9-prime-editing-scope","machine":"cas9","kind":"qa","prompt":"What share of known disease-associated genetic variants do the prime editing authors estimate it could in principle correct?","answer":"Up to 89 percent.","explanation":"Prime editing installs insertions, deletions and every point mutation class without double-strand breaks. The 89 percent is an author estimate of scope, not a measured correction rate.","section":"summary","topic":"debate","sources":["frontier:Writing without breaks","evidence:prime-editing-scope","ref:anzalone2019"],"tags":["prime-editing","frontier"],"difficulty":2,"url":"/machines/cas9#summary","cites":[{"source":"frontier:Writing without breaks","machine":"cas9","label":"Open question: Writing without breaks","section":"summary"},{"source":"evidence:prime-editing-scope","machine":"cas9","label":"Share of known pathogenic human variants that prime editing could in principle correct (Anzalone AV 2019)","section":"evidence","anchor":"ev-prime-editing-scope"},{"source":"ref:anzalone2019","machine":"cas9","label":"Anzalone et al.","section":"sources","anchor":"ref-anzalone2019","href":"https://doi.org/10.1038/s41586-019-1711-4"}]},{"id":"cas9-open-frontier","machine":"cas9","kind":"qa","prompt":"Which goal on the Cas9 frontier list is still only proposed rather than demonstrated?","answer":"Reliable in-place writing of whole genes or chromosomes.","explanation":"Today's editors change a few bases or insert short cassettes. Programmable writing of long sequences is still ahead.","section":"summary","topic":"debate","sources":["frontier:Programmable writing of long sequences"],"tags":["frontier","editing"],"difficulty":2,"url":"/machines/cas9#summary","cites":[{"source":"frontier:Programmable writing of long sequences","machine":"cas9","label":"Open question: Programmable writing of long sequences","section":"summary"}]},{"id":"cas9-guide-arms-protein","machine":"cas9","kind":"qa","prompt":"What has to happen before Cas9 can accept DNA at all?","answer":"The guide RNA must bind and turn the two lobes toward each other, forming the central channel.","explanation":"Apo Cas9 has no channel for DNA. Most of the rearrangement happens before any DNA arrives.","section":"mechanism","topic":"cycle","sources":["mechanism:The guide RNA arms the protein","ref:jinek2014"],"tags":["guide-rna","conformation"],"difficulty":2,"url":"/machines/cas9#mechanism","cites":[{"source":"mechanism:The guide RNA arms the protein","machine":"cas9","label":"Step: The guide RNA arms the protein","section":"mechanism"},{"source":"ref:jinek2014","machine":"cas9","label":"Jinek et al.","section":"sources","anchor":"ref-jinek2014","href":"https://doi.org/10.1126/science.1247997"}]},{"id":"cas9-skips-matches-without-pam","machine":"cas9","kind":"qa","prompt":"Why does Cas9 pass over a perfect sequence match that has no nearby PAM?","answer":"Binding and cleavage both need a short PAM next to the target.","explanation":"During the search, affinity for non-target DNA scales with PAM density, so the PAM is what the enzyme checks first.","section":"mechanism","topic":"cycle","sources":["mechanism:Cas9 reads PAMs while it scans","ref:sternberg2014"],"tags":["pam","search"],"difficulty":2,"url":"/machines/cas9#mechanism","cites":[{"source":"mechanism:Cas9 reads PAMs while it scans","machine":"cas9","label":"Step: Cas9 reads PAMs while it scans","section":"mechanism"},{"source":"ref:sternberg2014","machine":"cas9","label":"Sternberg et al.","section":"sources","anchor":"ref-sternberg2014","href":"https://doi.org/10.1038/nature13011"}]},{"id":"cas9-rloop-growth","machine":"cas9","kind":"cloze","prompt":"Strand separation in Cas9 starts at the {{PAM}}, and the RNA-DNA heteroduplex then grows away from there toward the far end of the target.","answer":"PAM","explanation":"That directional growth of base pairing builds the R-loop.","section":"mechanism","topic":"cycle","sources":["mechanism:The strands separate at the PAM","ref:jiang2016"],"tags":["r-loop","pam"],"difficulty":2,"url":"/machines/cas9#mechanism","cites":[{"source":"mechanism:The strands separate at the PAM","machine":"cas9","label":"Step: The strands separate at the PAM","section":"mechanism"},{"source":"ref:jiang2016","machine":"cas9","label":"Jiang et al.","section":"sources","anchor":"ref-jiang2016","href":"https://doi.org/10.1126/science.aad8282"}]},{"id":"cas9-first-checkpoint","machine":"cas9","kind":"qa","prompt":"What is the first checkpoint in the Cas9 cycle, the one that allows catalysis?","answer":"PAM recognition: PAM contacts trigger catalytic activity.","explanation":"Checking the PAM first means Cas9 wastes little time on sites it cannot cut.","section":"mechanism","topic":"cycle","sources":["mechanism:PAM recognition allows catalysis","ref:sternberg2014"],"tags":["pam","checkpoint"],"difficulty":2,"url":"/machines/cas9#mechanism","cites":[{"source":"mechanism:PAM recognition allows catalysis","machine":"cas9","label":"Step: PAM recognition allows catalysis","section":"mechanism"},{"source":"ref:sternberg2014","machine":"cas9","label":"Sternberg et al.","section":"sources","anchor":"ref-sternberg2014","href":"https://doi.org/10.1038/nature13011"}]},{"id":"cas9-final-checkpoint","machine":"cas9","kind":"qa","prompt":"Which step is the final specificity checkpoint of Cas9?","answer":"HNH docking into its activated conformation.","explanation":"Cleavage rate tracks how much HNH samples that conformation, and DNA binding is far less selective than cleavage.","section":"mechanism","topic":"cycle","sources":["mechanism:HNH docking decides","ref:sternberg2015"],"tags":["hnh","specificity"],"difficulty":3,"url":"/machines/cas9#mechanism","cites":[{"source":"mechanism:HNH docking decides","machine":"cas9","label":"Step: HNH docking decides","section":"mechanism"},{"source":"ref:sternberg2015","machine":"cas9","label":"Sternberg et al.","section":"sources","anchor":"ref-sternberg2015","href":"https://doi.org/10.1038/nature15544"}]},{"id":"cas9-concerted-cut","machine":"cas9","kind":"qa","prompt":"What makes the two nuclease domains of Cas9 fire together instead of one at a time?","answer":"Allosteric communication between them.","explanation":"HNH cuts the complementary strand and RuvC cuts the other, so the product is a double-strand break.","section":"mechanism","topic":"cycle","sources":["mechanism:Both strands cut together","ref:zhu2019"],"tags":["allostery","cleavage"],"difficulty":2,"url":"/machines/cas9#mechanism","cites":[{"source":"mechanism:Both strands cut together","machine":"cas9","label":"Step: Both strands cut together","section":"mechanism"},{"source":"ref:zhu2019","machine":"cas9","label":"Zhu et al.","section":"sources","anchor":"ref-zhu2019","href":"https://doi.org/10.1038/s41594-019-0258-2"}]},{"id":"cas9-ocean-infections","machine":"cas9","kind":"qa","prompt":"How many virus infections start in the ocean every second, the pressure that CRISPR immunity answers?","answer":"About 10^23 per second.","explanation":"Viruses that infect bacteria are the most numerous biological things on Earth, so bacteria have had to defend themselves for billions of years.","section":"story","topic":"numbers","sources":["fact:C2","story:summary","stop:planet","ref:suttle2007"],"tags":["phage","scale"],"difficulty":1,"url":"/machines/cas9#story","cites":[{"source":"fact:C2","machine":"cas9","label":"Virus infections in the ocean: 10^23 per second","section":"story"},{"source":"story:summary","machine":"cas9","label":"Big picture","section":"story"},{"source":"stop:planet","machine":"cas9","label":"Big picture: Planet of viruses","section":"story"},{"source":"ref:suttle2007","machine":"cas9","label":"Suttle 2007","section":"sources","href":"https://doi.org/10.1038/nrmicro1750"}]},{"id":"cas9-search-time","machine":"cas9","kind":"qa","prompt":"How long does one Cas9 need to find its target in E. coli?","answer":"About six hours.","explanation":"Measured by tracking one labelled dCas9. Because one copy is so slow, a cell keeps many copies searching.","section":"story","topic":"numbers","sources":["fact:C7","stop:search","ref:jones2017"],"tags":["search","kinetics"],"difficulty":2,"url":"/machines/cas9#story","cites":[{"source":"fact:C7","machine":"cas9","label":"Time for one Cas9 to find its target in E. coli: 6 hours","section":"story"},{"source":"stop:search","machine":"cas9","label":"Big picture: The search","section":"story"},{"source":"ref:jones2017","machine":"cas9","label":"Jones 2017","section":"sources","href":"https://doi.org/10.1126/science.aah7084"}]},{"id":"cas9-pam-spacing","machine":"cas9","kind":"cloze","prompt":"In random DNA an NGG PAM turns up about once every {{8}} base pairs, counting both strands.","answer":"8","explanation":"That is why the search has so many stops: about 10^6 PAM sites sit in one E. coli cell. In phage lambda DNA the spacing measured one PAM per 8.5 base pairs.","section":"story","topic":"numbers","sources":["fact:C6","ref:jones2017"],"tags":["pam","search"],"difficulty":2,"url":"/machines/cas9#story","cites":[{"source":"fact:C6","machine":"cas9","label":"Spacing of PAM sites in DNA: 8 base pairs between PAMs","section":"story"},{"source":"ref:jones2017","machine":"cas9","label":"Jones 2017","section":"sources","href":"https://doi.org/10.1126/science.aah7084"}]},{"id":"cas9-therapy-target","machine":"cas9","kind":"qa","prompt":"What does the approved Cas9 therapy cut in a patient's own blood stem cells?","answer":"The red-cell enhancer of the BCL11A gene.","explanation":"The red cells those stem cells make then produce fetal hemoglobin, which treats sickle cell disease and transfusion-dependent beta-thalassaemia.","section":"story","topic":"purpose","sources":["fact:C10","link:hemoglobin","ref:frangoul2021"],"tags":["medicine","hemoglobin"],"difficulty":2,"url":"/machines/cas9#story","cites":[{"source":"fact:C10","machine":"cas9","label":"What the approved therapy edits: BCL11A enhancer in blood stem cells","section":"story"},{"source":"link:hemoglobin","machine":"cas9","label":"Link to Hemoglobin","section":"story"},{"source":"ref:frangoul2021","machine":"cas9","label":"Frangoul 2021","section":"sources","href":"https://doi.org/10.1056/NEJMoa2031054"}]},{"id":"cas9-scissors-analogy-breaks","machine":"cas9","kind":"qa","prompt":"Calling Cas9 search-and-cut scissors gets one tool and one target right. What does it get wrong?","answer":"Scissors are aimed; Cas9 is not. It bumps into DNA at random, checks for a PAM and moves on.","explanation":"That random search is why finding one target takes hours.","section":"story","topic":"purpose","sources":["analogy:Search-and-cut scissors"],"tags":["analogy","search"],"difficulty":2,"url":"/machines/cas9#story","cites":[{"source":"analogy:Search-and-cut scissors","machine":"cas9","label":"Analogy: Search-and-cut scissors","section":"story"}]},{"id":"cas9-immune-memory-analogy-breaks","machine":"cas9","kind":"qa","prompt":"How is the bacterial CRISPR memory unlike our own immune memory?","answer":"It is written into the genome, so daughter cells inherit it.","explanation":"Both remember past infections and respond faster, but our antibody memory is not passed to our children.","section":"story","topic":"purpose","sources":["analogy:An immune memory","stop:attack"],"tags":["analogy","immunity"],"difficulty":2,"url":"/machines/cas9#story","cites":[{"source":"analogy:An immune memory","machine":"cas9","label":"Analogy: An immune memory","section":"story"},{"source":"stop:attack","machine":"cas9","label":"Big picture: Attack and memory","section":"story"}]},{"id":"cas9-residues-crystallised","machine":"cas9","kind":"cloze","prompt":"The ternary complex structure crystallised a full-length SpCas9 chain of {{1368}} residues.","answer":"1368","explanation":"X-ray crystallography at 2.5 A, with a 98-nucleotide single-guide RNA and a 23-nucleotide target DNA, anchors the sequence length in a primary structure study.","section":"evidence","topic":"numbers","sources":["evidence:spcas9-length-ternary-structure","stat:Residues","ref:nishimasu2014"],"tags":["structure","size"],"difficulty":1,"url":"/machines/cas9#evidence","cites":[{"source":"evidence:spcas9-length-ternary-structure","machine":"cas9","label":"Length of the SpCas9 polypeptide used for the ternary complex structure (Nishimasu H 2014)","section":"evidence","anchor":"ev-spcas9-length-ternary-structure"},{"source":"stat:Residues","machine":"cas9","label":"Key number: Residues","section":"summary"},{"source":"ref:nishimasu2014","machine":"cas9","label":"Nishimasu et al.","section":"sources","anchor":"ref-nishimasu2014","href":"https://doi.org/10.1016/j.cell.2014.02.001"}]},{"id":"cas9-single-mutant-nicks","machine":"cas9","kind":"qa","prompt":"What does a Cas9 carrying only one of the two active-site mutations do to DNA?","answer":"It nicks one strand instead of cutting both.","explanation":"Point mutants assayed on paired target sites offset by 4 base pairs showed which residue serves which strand: D10 for RuvC, H840 for HNH.","section":"evidence","topic":"numbers","sources":["evidence:catalytic-residues-nicking","stat:Catalytic residues"],"tags":["nickase","active-site"],"difficulty":2,"url":"/machines/cas9#evidence","cites":[{"source":"evidence:catalytic-residues-nicking","machine":"cas9","label":"Which active-site residue serves which strand (Nishimasu H 2014)","section":"evidence","anchor":"ev-catalytic-residues-nicking"},{"source":"stat:Catalytic residues","machine":"cas9","label":"Key number: Catalytic residues","section":"summary"}]},{"id":"cas9-binding-vs-cleavage-kd","machine":"cas9","kind":"qa","prompt":"Gel binding assays gave dCas9 a Kd of 0.80 nM on target. What was the Kd with 8 PAM-distal mismatches?","answer":"20 nM.","explanation":"A 25-fold spread in affinity sits beside cleavage that drops to undetectable, which is why binding is called far less selective than cleavage.","section":"evidence","topic":"numbers","sources":["evidence:binding-less-selective-than-cleavage","ref:sternberg2015"],"tags":["affinity","specificity"],"difficulty":3,"url":"/machines/cas9#evidence","cites":[{"source":"evidence:binding-less-selective-than-cleavage","machine":"cas9","label":"Binding affinity of catalytically dead Cas9 for on-target and mismatched DNA (Sternberg SH 2015)","section":"evidence","anchor":"ev-binding-less-selective-than-cleavage"},{"source":"ref:sternberg2015","machine":"cas9","label":"Sternberg et al.","section":"sources","anchor":"ref-sternberg2015","href":"https://doi.org/10.1038/nature15544"}]},{"id":"cas9-hnh-fret-method","machine":"cas9","kind":"qa","prompt":"How was the link between HNH conformation and cleavage rate in Cas9 measured?","answer":"Intramolecular FRET between dyes on REC1 and HNH, compared with cleavage rate constants on the same substrates.","explanation":"With enough PAM-distal mismatches the domain stays undocked and no cleavage is detected, which makes the conformational step the checkpoint.","section":"evidence","topic":"numbers","sources":["evidence:hnh-conformation-controls-cleavage","mechanism:HNH docking decides"],"tags":["fret","method"],"difficulty":3,"url":"/machines/cas9#evidence","cites":[{"source":"evidence:hnh-conformation-controls-cleavage","machine":"cas9","label":"Relation between HNH domain conformation and DNA cleavage rate (Sternberg SH 2015)","section":"evidence","anchor":"ev-hnh-conformation-controls-cleavage"},{"source":"mechanism:HNH docking decides","machine":"cas9","label":"Step: HNH docking decides","section":"mechanism"}]},{"id":"cas9-hnh-swing-distance","machine":"cas9","kind":"cloze","prompt":"Cryo-EM of the active Cas9 complex with magnesium shows the HNH domain travelling about {{34}} A to reach the target-strand scissile bond.","answer":"34","explanation":"Three states were resolved at about 3.3 A: pre-catalytic, post-catalytic and product. The motion is a translation with a rotation about a central axis.","section":"evidence","topic":"numbers","sources":["evidence:hnh-swing-to-cut-site","stat:HNH approach to the cut site","ref:zhu2019"],"tags":["cryo-em","hnh"],"difficulty":2,"url":"/machines/cas9#evidence","cites":[{"source":"evidence:hnh-swing-to-cut-site","machine":"cas9","label":"Distance the HNH domain travels to reach the target-strand scissile bond (Zhu X 2019)","section":"evidence","anchor":"ev-hnh-swing-to-cut-site"},{"source":"stat:HNH approach to the cut site","machine":"cas9","label":"Key number: HNH approach to the cut site","section":"summary"},{"source":"ref:zhu2019","machine":"cas9","label":"Zhu et al.","section":"sources","anchor":"ref-zhu2019","href":"https://doi.org/10.1038/s41594-019-0258-2"}]},{"id":"cas9-cut-position","machine":"cas9","kind":"qa","prompt":"Where does Cas9 cut the target strand, relative to the PAM?","answer":"Between the third and fourth nucleotide upstream of the PAM.","explanation":"Read from cryo-EM density across the cleavage site in the pre-catalytic, post-catalytic and product states.","section":"evidence","topic":"numbers","sources":["evidence:cut-position-upstream-of-pam","ref:zhu2019"],"tags":["cleavage","cryo-em"],"difficulty":2,"url":"/machines/cas9#evidence","cites":[{"source":"evidence:cut-position-upstream-of-pam","machine":"cas9","label":"Where the target strand is cut, relative to the PAM (Zhu X 2019)","section":"evidence","anchor":"ev-cut-position-upstream-of-pam"},{"source":"ref:zhu2019","machine":"cas9","label":"Zhu et al.","section":"sources","anchor":"ref-zhu2019","href":"https://doi.org/10.1038/s41594-019-0258-2"}]},{"id":"cas9-search-mode-dispute","machine":"cas9","kind":"qa","prompt":"Single-molecule imaging on DNA curtains found Cas9 reaching targets only by three-dimensional collisions. How did later FRET work qualify that?","answer":"It saw facilitated lateral diffusion carrying Cas9 from one PAM to a neighbouring one.","explanation":"Over short distances lateral diffusion competes with three-dimensional diffusion; no rate for it is confirmed from the abstract that is available.","section":"evidence","topic":"debate","sources":["evidence:lateral-diffusion-pam-search","evidence:nonspecific-dwell-times"],"tags":["search","single-molecule"],"difficulty":3,"url":"/machines/cas9#evidence","cites":[{"source":"evidence:lateral-diffusion-pam-search","machine":"cas9","label":"How Cas9 moves between nearby PAMs while searching (Globyte V 2019)","section":"evidence","anchor":"ev-lateral-diffusion-pam-search"},{"source":"evidence:nonspecific-dwell-times","machine":"cas9","label":"Lifetime of Cas9 on non-target DNA during the search (Sternberg SH 2014)","section":"evidence","anchor":"ev-nonspecific-dwell-times"}]},{"id":"shared-rotary-ion-motors","machine":"shared","kind":"qa","prompt":"Which two machines in the atlas are rotary motors driven by ions flowing across a membrane?","answer":"ATP synthase and the bacterial flagellar motor","explanation":"Both turn an inward flow of protons (or sodium ions) into rotation. Kinesin, dynein and myosin are walkers instead: they step along a filament and burn ATP.","section":"summary","topic":"purpose","sources":["flagellar-motor/link:atp-synthase","atp-synthase/machine:tagline","flagellar-motor/machine:tagline","kinesin/machine:tagline"],"tags":["rotary","ion-gradient","compare"],"difficulty":1,"url":"/learn","cites":[{"source":"flagellar-motor/link:atp-synthase","machine":"flagellar-motor","label":"Link to ATP synthase","section":"story"},{"source":"atp-synthase/machine:tagline","machine":"atp-synthase","label":"Summary","section":"summary"},{"source":"flagellar-motor/machine:tagline","machine":"flagellar-motor","label":"Summary","section":"summary"},{"source":"kinesin/machine:tagline","machine":"kinesin","label":"Summary","section":"summary"}]},{"id":"shared-flagellum-skips-atp","machine":"shared","kind":"qa","prompt":"ATP synthase and the flagellar motor both run on a proton gradient. What does the flagellar motor do differently with that energy?","answer":"It turns the proton flow straight into rotation, with no ATP in between.","explanation":"ATP synthase uses the same kind of gradient to make ATP, which other machines then spend.","section":"summary","topic":"purpose","sources":["atp-synthase/link:flagellar-motor","flagellar-motor/link:atp-synthase","flagellar-motor/machine:energy"],"tags":["energy","ion-gradient","rotary"],"difficulty":1,"url":"/learn","cites":[{"source":"atp-synthase/link:flagellar-motor","machine":"atp-synthase","label":"Link to Bacterial flagellar motor","section":"story"},{"source":"flagellar-motor/link:atp-synthase","machine":"flagellar-motor","label":"Link to ATP synthase","section":"story"},{"source":"flagellar-motor/machine:energy","machine":"flagellar-motor","label":"Summary","section":"summary"}]},{"id":"shared-sodium-motors","machine":"shared","kind":"cloze","prompt":"Besides protons, the polar flagellar motor of Vibrio and the ATP synthase of some bacteria run on a flow of {{sodium ions}}.","answer":"sodium ions","explanation":"Ion-driven rotary motors are not tied to protons. The fastest flagellar motor in the atlas, at 1,700 turns per second, is the Na+-driven motor of Vibrio alginolyticus.","section":"summary","topic":"purpose","sources":["flagellar-motor/machine:energy","atp-synthase/machine:energy","flagellar-motor/fact:F9"],"tags":["energy","sodium","ion-gradient"],"difficulty":2,"url":"/learn","cites":[{"source":"flagellar-motor/machine:energy","machine":"flagellar-motor","label":"Summary","section":"summary"},{"source":"atp-synthase/machine:energy","machine":"atp-synthase","label":"Summary","section":"summary"},{"source":"flagellar-motor/fact:F9","machine":"flagellar-motor","label":"Fastest flagellar motor: 1,700 turns per second","section":"story"}]},{"id":"shared-ribosome-gtp","machine":"shared","kind":"qa","prompt":"The motors in the atlas burn ATP. Which nucleotide do the ribosome's helpers EF-Tu and EF-G hydrolyse instead?","answer":"GTP","explanation":"The cell recharges GTP with a phosphate from ATP, using the enzyme nucleoside diphosphate kinase.","section":"summary","topic":"purpose","sources":["ribosome/machine:energy","ribosome/link:atp-synthase"],"tags":["energy","gtp"],"difficulty":1,"url":"/learn","cites":[{"source":"ribosome/machine:energy","machine":"ribosome","label":"Summary","section":"summary"},{"source":"ribosome/link:atp-synthase","machine":"ribosome","label":"Link to ATP synthase","section":"story"}]},{"id":"shared-no-fuel-machines","machine":"shared","kind":"qa","prompt":"Which two machines in the atlas spend no chemical fuel at all?","answer":"Cas9 and hemoglobin","explanation":"Cas9 relies on Mg2+ and the free energy of RNA-DNA base pairing. Hemoglobin relies on the oxygen gradient and its allosteric switch.","section":"summary","topic":"purpose","sources":["cas9/machine:energy","hemoglobin/machine:energy","cas9/link:atp-synthase"],"tags":["energy","compare"],"difficulty":2,"url":"/learn","cites":[{"source":"cas9/machine:energy","machine":"cas9","label":"Summary","section":"summary"},{"source":"hemoglobin/machine:energy","machine":"hemoglobin","label":"Summary","section":"summary"},{"source":"cas9/link:atp-synthase","machine":"cas9","label":"Link to ATP synthase","section":"story"}]},{"id":"shared-serca-reverse-trade","machine":"shared","kind":"qa","prompt":"ATP synthase uses an ion gradient to make ATP. What is the opposite trade that SERCA makes?","answer":"SERCA spends ATP to build an ion (calcium) gradient.","explanation":"SERCA burns one ATP to push two calcium ions out of the cytoplasm. ATP synthase can also run this way: with ATP and no gradient, it runs backward and pumps ions.","section":"summary","topic":"purpose","sources":["serca/link:atp-synthase","serca/machine:tagline","atp-synthase/machine:energy"],"tags":["energy","ion-gradient","pump"],"difficulty":2,"url":"/learn","cites":[{"source":"serca/link:atp-synthase","machine":"serca","label":"Link to ATP synthase","section":"story"},{"source":"serca/machine:tagline","machine":"serca","label":"Summary","section":"summary"},{"source":"atp-synthase/machine:energy","machine":"atp-synthase","label":"Summary","section":"summary"}]},{"id":"shared-power-stroke-trigger","machine":"shared","kind":"qa","prompt":"In both myosin and dynein, which nucleotide event, after the head binds its track, comes with the power stroke?","answer":"Phosphate release","explanation":"Myosin binds actin, releases phosphate and swings its lever; dynein rebinds the microtubule, phosphate leaves and the linker straightens. In kinesin, by contrast, ATP binding docks the neck linker, and that docking is the power stroke.","section":"summary","topic":"cycle","sources":["myosin/machine:summary","dynein/mechanism:Rebinding triggers the power stroke","kinesin/mechanism:ATP docks the neck linker"],"tags":["power-stroke","motors","compare"],"difficulty":3,"url":"/learn","cites":[{"source":"myosin/machine:summary","machine":"myosin","label":"Summary","section":"summary"},{"source":"dynein/mechanism:Rebinding triggers the power stroke","machine":"dynein","label":"Step: Rebinding triggers the power stroke","section":"mechanism"},{"source":"kinesin/mechanism:ATP docks the neck linker","machine":"kinesin","label":"Step: ATP docks the neck linker","section":"mechanism"}]},{"id":"shared-dynein-lever-unrelated","machine":"shared","kind":"qa","prompt":"Kinesin shares its catalytic fold with myosin. Which atlas motor also pulls with a lever-like part but belongs to an unrelated family?","answer":"Dynein (an AAA+ ring with a lever-like linker)","explanation":"Kinesin and myosin share almost no sequence, yet their cores share one fold and probably a common ancestor. Dynein's ring is built from six AAA+ modules instead.","section":"summary","topic":"parts","sources":["myosin/link:dynein","kinesin/evolution:Common core with myosin","dynein/evolution:AAA+ family member"],"tags":["evolution","motors","power-stroke"],"difficulty":2,"url":"/learn","cites":[{"source":"myosin/link:dynein","machine":"myosin","label":"Link to Dynein","section":"story"},{"source":"kinesin/evolution:Common core with myosin","machine":"kinesin","label":"Common core with myosin","section":"summary"},{"source":"dynein/evolution:AAA+ family member","machine":"dynein","label":"AAA+ family member","section":"summary"}]},{"id":"shared-flii-f1-relative","machine":"shared","kind":"qa","prompt":"Which part of the bacterial flagellum looks like the α and β subunits of the F1 head of ATP synthase?","answer":"The export ATPase FliI","explanation":"FliJ also looks like the coiled-coil of γ. This points to a common ancestor of the flagellar export machine and the rotary ATPases.","section":"summary","topic":"parts","sources":["flagellar-motor/evolution:Shared parts with ATP synthase","atp-synthase/evolution:Shared parts with the flagellum","atp-synthase/ref:imada2007"],"tags":["evolution","rotary"],"difficulty":2,"url":"/learn","cites":[{"source":"flagellar-motor/evolution:Shared parts with ATP synthase","machine":"flagellar-motor","label":"Shared parts with ATP synthase","section":"summary"},{"source":"atp-synthase/evolution:Shared parts with the flagellum","machine":"atp-synthase","label":"Shared parts with the flagellum","section":"summary"},{"source":"atp-synthase/ref:imada2007","machine":"atp-synthase","label":"Imada et al.","section":"sources","anchor":"ref-imada2007","href":"https://doi.org/10.1073/pnas.0608090104"}]},{"id":"shared-aaa-rings","machine":"shared","kind":"cloze","prompt":"Dynein's motor ring and the proteasome's substrate-pulling ring are both made of six {{AAA+}} ATPase units.","answer":"AAA+","explanation":"In dynein the six AAA+ modules sit in one heavy chain; in the proteasome they are six subunits, Rpt1 to Rpt6. The AAA+ fold forms active arrays in many cellular machines.","section":"summary","topic":"parts","sources":["dynein/evolution:AAA+ family member","proteasome/stat:ATPase subunits","dynein/stat:AAA+ domains in the ring","proteasome/evolution:AAA+ origin of the ATPase ring"],"tags":["evolution","aaa","atp"],"difficulty":2,"url":"/learn","cites":[{"source":"dynein/evolution:AAA+ family member","machine":"dynein","label":"AAA+ family member","section":"summary"},{"source":"proteasome/stat:ATPase subunits","machine":"proteasome","label":"Key number: ATPase subunits","section":"summary"},{"source":"dynein/stat:AAA+ domains in the ring","machine":"dynein","label":"Key number: AAA+ domains in the ring","section":"summary"},{"source":"proteasome/evolution:AAA+ origin of the ATPase ring","machine":"proteasome","label":"AAA+ origin of the ATPase ring","section":"summary"}]},{"id":"shared-xray-rna-catalyst","machine":"shared","kind":"qa","prompt":"How did Nissen and colleagues use X-ray crystallography to show that the ribosome's catalyst is RNA?","answer":"They soaked substrate mimics into crystals of the large subunit and found no protein within about 18 Å of the forming bond.","explanation":"The method needs crystals: the mimics were soaked into crystals of the archaeal 50S subunit. Only 23S rRNA touched the substrate analogues, so the ribosome is a ribozyme.","section":"evidence","topic":"numbers","sources":["ribosome/evidence:nissen2000-rna-catalyst","ribosome/ref:nissen2000"],"tags":["x-ray","methods","ribosome"],"difficulty":2,"url":"/learn","cites":[{"source":"ribosome/evidence:nissen2000-rna-catalyst","machine":"ribosome","label":"Distance from the forming peptide bond to the nearest protein (Nissen P 2000)","section":"evidence","anchor":"ev-nissen2000-rna-catalyst"},{"source":"ribosome/ref:nissen2000","machine":"ribosome","label":"Nissen et al.","section":"sources","anchor":"ref-nissen2000","href":"https://doi.org/10.1126/science.289.5481.920"}]},{"id":"shared-cryoem-crystal-contacts","machine":"shared","kind":"qa","prompt":"Cryo-EM showed liganded human hemoglobin turning further (about 22°) than the classic R crystal structure (15.0°). What did the cryo-EM molecules lack that crystal models have?","answer":"Crystal contacts","explanation":"Cryo-EM images single molecules in solution-like conditions. Free of crystal contacts, the liganded tetramer sat almost on top of the R2 crystal form, so a single R state is a simplification.","section":"evidence","topic":"debate","sources":["hemoglobin/evidence:liganded-human-hb-closer-to-r2","hemoglobin/evidence:quaternary-rotation-t-to-r"],"tags":["cryo-em","x-ray","methods"],"difficulty":2,"url":"/learn","cites":[{"source":"hemoglobin/evidence:liganded-human-hb-closer-to-r2","machine":"hemoglobin","label":"Quaternary position of liganded human hemoglobin measured by cryo-EM in solution-like conditions (Takahashi K 2024)","section":"evidence","anchor":"ev-liganded-human-hb-closer-to-r2"},{"source":"hemoglobin/evidence:quaternary-rotation-t-to-r","machine":"hemoglobin","label":"Rotation of one alpha-beta dimer against the other, deoxy human hemoglobin to the R state (Takahashi K 2024)","section":"evidence","anchor":"ev-quaternary-rotation-t-to-r"}]},{"id":"shared-cryoem-sorting-states","machine":"shared","kind":"qa","prompt":"Cryo-EM studies of the proteasome, dynein and GroEL each found several cycle states in one sample. How?","answer":"They froze the machines while they worked and sorted the particle images into separate classes (states).","explanation":"Dong and colleagues sorted about 3.6 million proteasome images into seven states; Chai and colleagues found eight major dynein states.","section":"evidence","topic":"cycle","sources":["proteasome/evidence:seven-substrate-engaged-states","dynein/evidence:chai2025-cycle-states","groel/evidence:apical-domain-tilt"],"tags":["cryo-em","methods"],"difficulty":2,"url":"/learn","cites":[{"source":"proteasome/evidence:seven-substrate-engaged-states","machine":"proteasome","label":"Distinct conformations resolved from one substrate-engaged sample (Dong Y 2019)","section":"evidence","anchor":"ev-seven-substrate-engaged-states"},{"source":"dynein/evidence:chai2025-cycle-states","machine":"dynein","label":"Conformational states of the human dynein-1 motor during its cycle (Chai P 2025)","section":"evidence","anchor":"ev-chai2025-cycle-states"},{"source":"groel/evidence:apical-domain-tilt","machine":"groel","label":"First domain movements after ATP binding (Clare DK 2012)","section":"evidence","anchor":"ev-apical-domain-tilt"}]},{"id":"shared-time-resolved-myosin","machine":"shared","kind":"qa","prompt":"How did Klebl and colleagues use cryo-EM to catch myosin-5 both before and after its power stroke?","answer":"They mixed myosin-ADP-Pi with actin and plunge-froze the mix 10 or 120 ms later (time-resolved cryo-EM).","explanation":"The frozen sample held primed and post-stroke motors on the same filament. The lever swung about 93°, mostly along the actin axis.","section":"evidence","topic":"cycle","sources":["myosin/evidence:lever-swing-klebl2025","myosin/mechanism:The power stroke","myosin/ref:klebl2025"],"tags":["cryo-em","power-stroke","methods"],"difficulty":3,"url":"/learn","cites":[{"source":"myosin/evidence:lever-swing-klebl2025","machine":"myosin","label":"Lever swing of myosin-5 on actin, primed to post-power stroke (Klebl DP 2025)","section":"evidence","anchor":"ev-lever-swing-klebl2025"},{"source":"myosin/mechanism:The power stroke","machine":"myosin","label":"Step: The power stroke","section":"mechanism"},{"source":"myosin/ref:klebl2025","machine":"myosin","label":"Klebl et al.","section":"sources","anchor":"ref-klebl2025","href":"https://doi.org/10.1038/s41586-025-08876-5"}]},{"id":"shared-c8-proton-cost","machine":"shared","kind":"cloze","prompt":"The X-ray structure of the bovine F1–c-ring showed 8 c subunits, so animal ATP synthase spends about {{2.7}} protons per ATP.","answer":"2.7","explanation":"Eight protons turn the ring once and one turn makes three ATP: 8/3 ≈ 2.7. Larger c-rings (up to c15) cost more protons per ATP.","section":"evidence","topic":"numbers","sources":["atp-synthase/evidence:c8-ring-bovine","atp-synthase/stat:H+ per ATP in animals","atp-synthase/ref:watt2010"],"tags":["x-ray","energy","rotary"],"difficulty":2,"url":"/learn","cites":[{"source":"atp-synthase/evidence:c8-ring-bovine","machine":"atp-synthase","label":"Number of c subunits in the mammalian c-ring (Watt IN 2010)","section":"evidence","anchor":"ev-c8-ring-bovine"},{"source":"atp-synthase/stat:H+ per ATP in animals","machine":"atp-synthase","label":"Key number: H+ per ATP in animals","section":"summary"},{"source":"atp-synthase/ref:watt2010","machine":"atp-synthase","label":"Watt et al.","section":"sources","anchor":"ref-watt2010","href":"https://doi.org/10.1073/pnas.1011099107"}]},{"id":"shared-step-sizes","machine":"shared","kind":"qa","prompt":"Kinesin-1 steps 8 nm and yeast dynein most often steps 8 nm. About how far does myosin-5 step per ATP on actin?","answer":"About 37 nm","explanation":"The myosin-5 step matches the 36 nm pseudo-repeat of actin. Kinesin's 8 nm is the length of one tubulin dimer.","section":"story","topic":"numbers","sources":["myosin/stat:Myosin-5 step","kinesin/stat:Step size","dynein/stat:Most frequent step","myosin/ref:yildiz2003"],"tags":["scale","motors","walkers"],"difficulty":2,"url":"/learn","cites":[{"source":"myosin/stat:Myosin-5 step","machine":"myosin","label":"Key number: Myosin-5 step","section":"summary"},{"source":"kinesin/stat:Step size","machine":"kinesin","label":"Key number: Step size","section":"summary"},{"source":"dynein/stat:Most frequent step","machine":"dynein","label":"Key number: Most frequent step","section":"summary"},{"source":"myosin/ref:yildiz2003","machine":"myosin","label":"Yildiz et al.","section":"sources","anchor":"ref-yildiz2003","href":"https://doi.org/10.1126/science.1084398"}]},{"id":"shared-fastest-rotor","machine":"shared","kind":"qa","prompt":"Which motor is the fastest rotary motor in the atlas, at about 1,700 turns per second, and what ion drives it?","answer":"The flagellar motor of Vibrio alginolyticus, driven by sodium ions","explanation":"For comparison, E. coli flagellar motors reach about 300–350 turns per second with no load, and ATP synthase in mitochondria is estimated at about 100 turns per second.","section":"story","topic":"numbers","sources":["flagellar-motor/fact:F9","flagellar-motor/fact:s-speed","atp-synthase/fact:A14"],"tags":["scale","rotary","sodium"],"difficulty":2,"url":"/learn","cites":[{"source":"flagellar-motor/fact:F9","machine":"flagellar-motor","label":"Fastest flagellar motor: 1,700 turns per second","section":"story"},{"source":"flagellar-motor/fact:s-speed","machine":"flagellar-motor","label":"Top speed with no load: 300–350 turns per second","section":"story"},{"source":"atp-synthase/fact:A14","machine":"atp-synthase","label":"Turn rate in mitochondria: 100 turns per second","section":"story"}]},{"id":"shared-largest-mass","machine":"shared","kind":"qa","prompt":"Which machine in the atlas has the largest listed mass?","answer":"The flagellar motor (Salmonella motor–hook model, about 14,881 kDa)","explanation":"That model has no stator units and only the proximal hook. The human 26S proteasome is about 2.5 MDa and the E. coli 70S ribosome about 2.2 MDa; hemoglobin is 64.5 kDa.","section":"story","topic":"numbers","sources":["flagellar-motor/machine:size","proteasome/machine:size","ribosome/machine:size","hemoglobin/machine:size"],"tags":["scale","size","compare"],"difficulty":2,"url":"/learn","cites":[{"source":"flagellar-motor/machine:size","machine":"flagellar-motor","label":"Summary","section":"summary"},{"source":"proteasome/machine:size","machine":"proteasome","label":"Summary","section":"summary"},{"source":"ribosome/machine:size","machine":"ribosome","label":"Summary","section":"summary"},{"source":"hemoglobin/machine:size","machine":"hemoglobin","label":"Summary","section":"summary"}]},{"id":"shared-make-vs-destroy","machine":"shared","kind":"qa","prompt":"In one mouse L929 cell, how does the proteasome's rate of destroying proteins compare with the ribosomes' rate of making them?","answer":"About half: 2 × 10^6 destroyed per minute against 4 × 10^6 made per minute","explanation":"Together the two machines set protein levels, and the recycled amino acids feed the ribosome.","section":"story","topic":"numbers","sources":["ribosome/link:proteasome","proteasome/fact:P12","ribosome/fact:R5","proteasome/link:ribosome"],"tags":["scale","counts","compare"],"difficulty":2,"url":"/learn","cites":[{"source":"ribosome/link:proteasome","machine":"ribosome","label":"Link to Proteasome","section":"story"},{"source":"proteasome/fact:P12","machine":"proteasome","label":"Proteins destroyed per minute in one mouse cell: 2 × 10^6 per minute","section":"story"},{"source":"ribosome/fact:R5","machine":"ribosome","label":"Proteins made per minute in one mouse cell: 4 × 10^6 per minute","section":"story"},{"source":"proteasome/link:ribosome","machine":"proteasome","label":"Link to Ribosome","section":"story"}]},{"id":"shared-atp-body-weight","machine":"shared","kind":"cloze","prompt":"Every ATP-burning machine in the atlas draws on ATP synthase, and a human body remakes about {{one body weight}} of ATP per day.","answer":"one body weight","explanation":"An upper estimate from oxygen use: 54–68 kg per day for a college student. The links show kinesin, myosin, dynein, SERCA, GroEL and the proteasome all spending this ATP.","section":"story","topic":"numbers","sources":["atp-synthase/fact:A2","atp-synthase/link:kinesin","atp-synthase/link:serca"],"tags":["scale","atp","energy"],"difficulty":1,"url":"/learn","cites":[{"source":"atp-synthase/fact:A2","machine":"atp-synthase","label":"ATP remade per day: 1 body weight","section":"story"},{"source":"atp-synthase/link:kinesin","machine":"atp-synthase","label":"Link to Kinesin","section":"story"},{"source":"atp-synthase/link:serca","machine":"atp-synthase","label":"Link to SERCA calcium pump","section":"story"}]}]}