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- EMDB-31846: Nucleotide-free V/A-ATPase from Thermus thermophilus, state3 -

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Basic information

Entry
Database: EMDB / ID: EMD-31846
TitleNucleotide-free V/A-ATPase from Thermus thermophilus, state3
Map dataNucleotide-free V/A-ATPase from Thermus thermophilus, state3, postprocessed map.
Sample
  • Complex: Nucleotide-free V/A-ATPase from Thermus thermophilus, state3
    • Protein or peptide: A subunit of V/A-ATPase from Thermus thremophilus
    • Protein or peptide: B subunit of V/A-ATPase from Thermus thermophilus
    • Protein or peptide: D subunit of V/A-ATPase from Thermus thermophilus
    • Protein or peptide: F subunit of V/A-ATPase from Thermus thermophilus
    • Protein or peptide: E subunit of V/A-ATPase from Thermus thermophilus
    • Protein or peptide: G subunit of V/A-ATPase from Thermus thermophilus
    • Protein or peptide: a subunit of V/A-ATPase from Thermus thermophilus
    • Protein or peptide: d subunit of V/A-ATPase from Thermus thermophilus
    • Protein or peptide: c subunit of V/A-ATPase from Thermus thermophilus
Biological speciesThermus thermophilus HB8 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.3 Å
AuthorsKishikawa J / Nakanishi A / Nakano A / Saeki S / Furuta A / Kato T / Mitsuoka K / Yokoyama K
Funding support Japan, 6 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)20H03231 Japan
Japan Society for the Promotion of Science (JSPS)20K06514 Japan
Japan Society for the Promotion of Science (JSPS)20J00162 Japan
Japan Agency for Medical Research and Development (AMED)JP17am0101001 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)12024046 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)20215008 Japan
CitationJournal: Nat Commun / Year: 2022
Title: Structural snapshots of V/A-ATPase reveal the rotary catalytic mechanism of rotary ATPases.
Authors: J Kishikawa / A Nakanishi / A Nakano / S Saeki / A Furuta / T Kato / K Mistuoka / K Yokoyama /
Abstract: V/A-ATPase is a motor protein that shares a common rotary catalytic mechanism with FF ATP synthase. When powered by ATP hydrolysis, the V domain rotates the central rotor against the AB hexamer, ...V/A-ATPase is a motor protein that shares a common rotary catalytic mechanism with FF ATP synthase. When powered by ATP hydrolysis, the V domain rotates the central rotor against the AB hexamer, composed of three catalytic AB dimers adopting different conformations (AB, AB, and AB). Here, we report the atomic models of 18 catalytic intermediates of the V domain of V/A-ATPase under different reaction conditions, determined by single particle cryo-EM. The models reveal that the rotor does not rotate immediately after binding of ATP to the V. Instead, three events proceed simultaneously with the 120˚ rotation of the shaft: hydrolysis of ATP in AB, zipper movement in AB by the binding ATP, and unzipper movement in AB with release of both ADP and Pi. This indicates the unidirectional rotation of V/A-ATPase by a ratchet-like mechanism owing to ATP hydrolysis in AB, rather than the power stroke model proposed previously for F-ATPase.
History
DepositionAug 30, 2021-
Header (metadata) releaseJul 13, 2022-
Map releaseJul 13, 2022-
UpdateJul 13, 2022-
Current statusJul 13, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_31846.png

Downloads & links

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Map

FileDownload / File: emd_31846.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNucleotide-free V/A-ATPase from Thermus thermophilus, state3, postprocessed map.
Voxel sizeX=Y=Z: 0.88 Å
Density
Contour LevelBy AUTHOR: 0.005
Minimum - Maximum-0.008493024 - 0.017552614
Average (Standard dev.)5.4970147e-05 (±0.0010260663)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions450450450
Spacing450450450
CellA=B=C: 396.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_31846_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Nucleotide-free V/A-ATPase from Thermus thermophilus, state3, halfmap2....

Fileemd_31846_half_map_1.map
AnnotationNucleotide-free V/A-ATPase from Thermus thermophilus, state3, halfmap2.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Nucleotide-free V/A-ATPase from Thermus thermophilus, state3, halfmap1....

Fileemd_31846_half_map_2.map
AnnotationNucleotide-free V/A-ATPase from Thermus thermophilus, state3, halfmap1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Nucleotide-free V/A-ATPase from Thermus thermophilus, state3

EntireName: Nucleotide-free V/A-ATPase from Thermus thermophilus, state3
Components
  • Complex: Nucleotide-free V/A-ATPase from Thermus thermophilus, state3
    • Protein or peptide: A subunit of V/A-ATPase from Thermus thremophilus
    • Protein or peptide: B subunit of V/A-ATPase from Thermus thermophilus
    • Protein or peptide: D subunit of V/A-ATPase from Thermus thermophilus
    • Protein or peptide: F subunit of V/A-ATPase from Thermus thermophilus
    • Protein or peptide: E subunit of V/A-ATPase from Thermus thermophilus
    • Protein or peptide: G subunit of V/A-ATPase from Thermus thermophilus
    • Protein or peptide: a subunit of V/A-ATPase from Thermus thermophilus
    • Protein or peptide: d subunit of V/A-ATPase from Thermus thermophilus
    • Protein or peptide: c subunit of V/A-ATPase from Thermus thermophilus

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Supramolecule #1: Nucleotide-free V/A-ATPase from Thermus thermophilus, state3

SupramoleculeName: Nucleotide-free V/A-ATPase from Thermus thermophilus, state3
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 700 KDa

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Macromolecule #1: A subunit of V/A-ATPase from Thermus thremophilus

MacromoleculeName: A subunit of V/A-ATPase from Thermus thremophilus / type: protein_or_peptide / ID: 1
Details: S232A and T235S are mutated residues for nucleotide binding.
Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
SequenceString: MIQGVIQKIA G PAVIAKGM LGARMYDICK VG EEGLVGE IIRLDGDTAF VQV YEDTSG LKVGEPVVST GLPL AVELG PGMLNGIYDG IQRPL ERIR EKTGIYITRG VVVHAL DRE KKWAWTPMVK PGDEVRG GM VLGTVPEFGF THKILVPP D VRGRVKEVKP ...String:
MIQGVIQKIA G PAVIAKGM LGARMYDICK VG EEGLVGE IIRLDGDTAF VQV YEDTSG LKVGEPVVST GLPL AVELG PGMLNGIYDG IQRPL ERIR EKTGIYITRG VVVHAL DRE KKWAWTPMVK PGDEVRG GM VLGTVPEFGF THKILVPP D VRGRVKEVKP AGEYTVEEP VVVLEDGTEL KMYHTWPVRR ARPVQRKLD PNTPFLTGMR I LDVLFPVA MGGTAAIPGP FG AGKSVTQ QSLAKWSNAD VVV YVGCGE RGNEMTDVLV EFPE LTDPK TGGPLMHRTV LIANT SNMP VAAREASIYV GVTIAE YFR DQGFSVALMA DSTSRWA EA LREISSRLEE MPAEEGYP P YLAARLAAFY ERAGKVITL GGEEGAVTIV GAVSPPGGDM SEPVTQSTL RIVGAFWRLD A SLAFRRHF PAINWNGSYS LF TSALDPW YRENVAEDYP ELR DAISEL LQREAGLQEI VQLV GPDAL QDAERLVIEV GRIIR EDFL QQNAYHEVDA YCSMKK AYG IMKMILAFYK EAEAAIK RG VSIDEILQLP VLERIGRA R YVSEEEFPAY FEEAMKEIQ GAFKALA

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Macromolecule #2: B subunit of V/A-ATPase from Thermus thermophilus

MacromoleculeName: B subunit of V/A-ATPase from Thermus thermophilus / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
SequenceString: MDLLKKEYTG ITYISGPLLF VENAKDLAYG AIVDIKDGTG RVRGGQVIEV SEEYAVIQVF EETTGLDLAT TSVSLVEDVA RLGVSKEMLG RRFNGIGKPI DGLPPITPEK RLPITGLPLN PVARRKPEQF IQTGISTIDV MNTLVRGQKL PIFSGSGLPA NEIAAQIARQ ...String:
MDLLKKEYTG ITYISGPLLF VENAKDLAYG AIVDIKDGTG RVRGGQVIEV SEEYAVIQVF EETTGLDLAT TSVSLVEDVA RLGVSKEMLG RRFNGIGKPI DGLPPITPEK RLPITGLPLN PVARRKPEQF IQTGISTIDV MNTLVRGQKL PIFSGSGLPA NEIAAQIARQ ATVRPDLSGE GEKEEPFAVV FAAMGITQRE LSYFIQEFER TGALSRSVLF LNKADDPTIE RILTPRMALT VAEYLAFEHD YHVLVILTDM TNYCEALREI GAAREEIPGR RGYPGYMYTD LATIYERAGV VEGKKGSVTQ IPILSMPDDD RTHPIPDLTG YITEGQIQLS RELHRKGIYP PIDPLPSLSR LMNNGVGKGK TREDHKQVSD QLYSAYANGV DIRKLVAIIG EDALTENDRR YLQFADAFER FFINQGQQNR SIEESLQIAW ALLSMLPQGE LKRISKDHIG KYYGQKLEEI WGAPQALD

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Macromolecule #3: D subunit of V/A-ATPase from Thermus thermophilus

MacromoleculeName: D subunit of V/A-ATPase from Thermus thermophilus / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
SequenceString: MSQVSPTRMN LLQRRGQLRL AQKGVDLLKK KRDALVAEFF GLVREAMEAR KALDQAAKEA YAALLLAQAF DGPEVVAGAA LGVPPLEGVE AEVENVWGSK VPRLKATFPD GALLSPVGTP AYTLEASRAF RRYAEALIRV ANTETRLKKI GEEIKKTTRR VNALEQVVIP ...String:
MSQVSPTRMN LLQRRGQLRL AQKGVDLLKK KRDALVAEFF GLVREAMEAR KALDQAAKEA YAALLLAQAF DGPEVVAGAA LGVPPLEGVE AEVENVWGSK VPRLKATFPD GALLSPVGTP AYTLEASRAF RRYAEALIRV ANTETRLKKI GEEIKKTTRR VNALEQVVIP GIRAQIRFIQ QVLEQRERED TFRLKRIKGK IEAREAEEEG GRPNPQVEIG AGL

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Macromolecule #4: F subunit of V/A-ATPase from Thermus thermophilus

MacromoleculeName: F subunit of V/A-ATPase from Thermus thermophilus / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
SequenceString:
MAVIADPETA QGFRLAGLEG YGASSAEEAQ SLLETLVERG GYALVAVDEA LLPDPERAVE RLMRGRDLPV LLPIAGLKEA FQGHDVEGYM RELVRKTIGF DIKL

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Macromolecule #5: E subunit of V/A-ATPase from Thermus thermophilus

MacromoleculeName: E subunit of V/A-ATPase from Thermus thermophilus / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
SequenceString:
MSKLEAILSQ EVEAEIQALL QEAEAKAEAV KREAEEKAKA LLQARERALE AQYRAALRRA ESAGELLVAT ARTQARGEVL EEVRRRVREA LEALPQKPEW PEVVRKLALE ALEALPGAKA LVANPEDLPH LEALARERGV ELQAEPALRL GVRAVGAEGK TQVENSLLAR LDRAWDALSS KVAQALWG

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Macromolecule #6: G subunit of V/A-ATPase from Thermus thermophilus

MacromoleculeName: G subunit of V/A-ATPase from Thermus thermophilus / type: protein_or_peptide / ID: 6 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
SequenceString:
MTGGLVLNAI SRAGGAMGGL GLIKSLAEKE KQLLERLEAA KKEAEERVKR AEAEAKALLE EAEAKAKALE AQYRERERAE TEALLARYRE RAEAEAKAVR EKAMARLDEA VALVLKEVLP

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Macromolecule #7: a subunit of V/A-ATPase from Thermus thermophilus

MacromoleculeName: a subunit of V/A-ATPase from Thermus thermophilus / type: protein_or_peptide / ID: 7 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
SequenceString: MIAPMEKLVL AGPKGRAKEL LQSLQQAGVV HLETLRPEAL SAYQLSPEER AELRRWEAVS AGAEHTLSLL GLEAEPARPF PEGLEAAEKA LSPIQAHAEG LTRQKQELEE ELALAQAYLE PLERLAALAH GLDKSPFLRV IPFLLTEKEL PLVEEALRKA LEDRYLLAHE ...String:
MIAPMEKLVL AGPKGRAKEL LQSLQQAGVV HLETLRPEAL SAYQLSPEER AELRRWEAVS AGAEHTLSLL GLEAEPARPF PEGLEAAEKA LSPIQAHAEG LTRQKQELEE ELALAQAYLE PLERLAALAH GLDKSPFLRV IPFLLTEKEL PLVEEALRKA LEDRYLLAHE AYAGGVAALV VVHRKEVDQA KAALSRAGVA ELRLPGALGE LPLSEAARRL KERAEAAPRE LSEVRQHLAK LARESASTLQ SLWTRAQDEV ARLKALEELA SGRFGFALLG YVPVKAKPKV EEALARHKES VVYAFEPVDE HHEADRIPVV LDNPPWAKPF ELLVSFLNTP KYGTFDPTPV VPVFFPFWFG MIVGDIGYAL LFYLVGRWLS GYVKRNEPLV IDLFALKLKP QVIGKLVHIL NWMVFWTVVW GVIYGEFFGT FLEHLGVFGT PEHPGLIPIL IHRIDTAKTA NLLILLSVAF GVVLVFFGLA LRAYLGLKHR HMAHFWEGVG YLGGLVGVLA LAASYLGNLQ AGWLQGLMYL GFGVFLLAVL MSRIWLMIPE IFTQAGHILS HIRIYAVGAA GGILAGLLTD VGFALAERLG LLGVLLGLLV AGVLHLLILL LTTLGHMLQP IRLLWVEFFT KFGFYEENGR PYRPFKSVRE AQ

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Macromolecule #8: d subunit of V/A-ATPase from Thermus thermophilus

MacromoleculeName: d subunit of V/A-ATPase from Thermus thermophilus / type: protein_or_peptide / ID: 8 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
SequenceString: MADDFAYLNA RVRVRRGTLL KESFFQEALD LSFADFLRLL SETVYGGELA GQGLPDVDRA VLRTQAKLVG DLPRLVTGEA REAVRLLLLR NDLHNLQALL RAKATGRPFE EVLLLPGTLR EEVWRQAYEA QDPAGMAQVL AVPGHPLARA LRAVLRETQD LARVEALLAK ...String:
MADDFAYLNA RVRVRRGTLL KESFFQEALD LSFADFLRLL SETVYGGELA GQGLPDVDRA VLRTQAKLVG DLPRLVTGEA REAVRLLLLR NDLHNLQALL RAKATGRPFE EVLLLPGTLR EEVWRQAYEA QDPAGMAQVL AVPGHPLARA LRAVLRETQD LARVEALLAK RFFEDVAKAA KGLDQPALRD YLALEVDAEN LRTAFKLQGS GLAPDAFFLK GGRFVDRVRF ARLMEGDYAV LDELSGTPFS GLSGVRDLKA LERGLRCVLL KEAKKGVQDP LGVGLVLAYV KEREWEAVRL RLLARRAYFG LPRAQVEEEV VCP

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Macromolecule #9: c subunit of V/A-ATPase from Thermus thermophilus

MacromoleculeName: c subunit of V/A-ATPase from Thermus thermophilus / type: protein_or_peptide / ID: 9 / Details: His-tag for purification at C-terminal / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
SequenceString:
MKKLLVTVLL AVFGALAFAA EEAAASGGLD RGLIAVGMGL AVGLAALGTG VAQARIGAAG VGAIAEDRSN FGTALIFLLL PETLVIFGLL IAFILNGRLH HH

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: MOLYBDENUM / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.045 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average exposure time: 5.0 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4354341
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Startup modelType of model: OTHER / Details: The map obtaind in previous study
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 6.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 5188
FSC plot (resolution estimation)

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