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- EMDB-30014: V1-ATPase built from cryo-EM map of V/A-ATPase from Thermus therm... -

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Basic information

Entry
Database: EMDB / ID: EMD-30014
TitleV1-ATPase built from cryo-EM map of V/A-ATPase from Thermus thermophilus.
Map dataCryo-EM maps of V/A-ATPase from Thermus thermophilus. Soluble domain including V1, d, two EG stalks, and N-terminal domain of a-subunit.
Sample
  • Complex: Soluble domain of V/A-ATPase including V1, d, two EG stalks, and N-terminal of a-subunit
    • Protein or peptide: V-type ATP synthase alpha chain
    • Protein or peptide: V-type ATP synthase beta chain
    • Protein or peptide: V-type ATP synthase subunit D
    • Protein or peptide: V-type ATP synthase subunit F
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
Function / homology
Function and homology information


proton-transporting ATP synthase complex / proton motive force-driven plasma membrane ATP synthesis / H+-transporting two-sector ATPase / proton-transporting ATPase activity, rotational mechanism / proton-transporting ATP synthase activity, rotational mechanism / ATP binding
Similarity search - Function
ATPase, V1 complex, subunit F, bacterial/archaeal / ATPase, V1 complex, subunit D / ATPase, V1 complex, subunit F / ATPase, V1 complex, subunit F superfamily / ATP synthase subunit D / ATP synthase (F/14-kDa) subunit / V-type ATP synthase regulatory subunit B/beta / V-type ATP synthase catalytic alpha chain / ATPsynthase alpha/beta subunit, N-terminal extension / ATPsynthase alpha/beta subunit N-term extension ...ATPase, V1 complex, subunit F, bacterial/archaeal / ATPase, V1 complex, subunit D / ATPase, V1 complex, subunit F / ATPase, V1 complex, subunit F superfamily / ATP synthase subunit D / ATP synthase (F/14-kDa) subunit / V-type ATP synthase regulatory subunit B/beta / V-type ATP synthase catalytic alpha chain / ATPsynthase alpha/beta subunit, N-terminal extension / ATPsynthase alpha/beta subunit N-term extension / ATPase, F1/V1 complex, beta/alpha subunit, C-terminal / ATP synthase subunit alpha, N-terminal domain-like superfamily / ATPase, F1/V1/A1 complex, alpha/beta subunit, N-terminal domain superfamily / ATPase, F1/V1/A1 complex, alpha/beta subunit, N-terminal domain / ATP synthase alpha/beta family, beta-barrel domain / ATPase, alpha/beta subunit, nucleotide-binding domain, active site / ATP synthase alpha and beta subunits signature. / ATPase, F1/V1/A1 complex, alpha/beta subunit, nucleotide-binding domain / ATP synthase alpha/beta family, nucleotide-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
V-type ATP synthase subunit D / V-type ATP synthase subunit F / V-type ATP synthase alpha chain / V-type ATP synthase beta chain
Similarity search - Component
Biological speciesThermus thermophilus HB8 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsKishikawa J / Nakanishi A / Furuta A / Kato T / Namba K / Tamakoshi M / Mitsuoka K / Yokoyama K
Funding support Japan, 3 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)17H03648 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)12024046 Japan
Japan Agency for Medical Research and Development (AMED)JP17am0101001 Japan
CitationJournal: Elife / Year: 2020
Title: Mechanical inhibition of isolated V from V/A-ATPase for proton conductance.
Authors: Jun-Ichi Kishikawa / Atsuko Nakanishi / Aya Furuta / Takayuki Kato / Keiichi Namba / Masatada Tamakoshi / Kaoru Mitsuoka / Ken Yokoyama /
Abstract: V-ATPase is an energy converting enzyme, coupling ATP hydrolysis/synthesis in the hydrophilic V domain, with proton flow through the V membrane domain, via rotation of the central rotor complex ...V-ATPase is an energy converting enzyme, coupling ATP hydrolysis/synthesis in the hydrophilic V domain, with proton flow through the V membrane domain, via rotation of the central rotor complex relative to the surrounding stator apparatus. Upon dissociation from the V domain, the V domain of the eukaryotic V-ATPase can adopt a physiologically relevant auto-inhibited form in which proton conductance through the V domain is prevented, however the molecular mechanism of this inhibition is not fully understood. Using cryo-electron microscopy, we determined the structure of both the V/A-ATPase and isolated V at near-atomic resolution, respectively. These structures clarify how the isolated V domain adopts the auto-inhibited form and how the complex prevents formation of the inhibited V form.
History
DepositionFeb 13, 2020-
Header (metadata) releaseSep 9, 2020-
Map releaseSep 9, 2020-
UpdateSep 30, 2020-
Current statusSep 30, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.068
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.068
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ly8
  • Surface level: 0.068
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6ly8
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30014.png

Downloads & links

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Map

FileDownload / File: emd_30014.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM maps of V/A-ATPase from Thermus thermophilus. Soluble domain including V1, d, two EG stalks, and N-terminal domain of a-subunit.
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.068 / Movie #1: 0.068
Minimum - Maximum-0.36541933 - 0.51166797
Average (Standard dev.)0.000580391 (±0.010872319)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 374.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z340340340
origin x/y/z0.0000.0000.000
length x/y/z374.000374.000374.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ480480480
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS340340340
D min/max/mean-0.3650.5120.001

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Supplemental data

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Sample components

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Entire : Soluble domain of V/A-ATPase including V1, d, two EG stalks, and ...

EntireName: Soluble domain of V/A-ATPase including V1, d, two EG stalks, and N-terminal of a-subunit
Components
  • Complex: Soluble domain of V/A-ATPase including V1, d, two EG stalks, and N-terminal of a-subunit
    • Protein or peptide: V-type ATP synthase alpha chain
    • Protein or peptide: V-type ATP synthase beta chain
    • Protein or peptide: V-type ATP synthase subunit D
    • Protein or peptide: V-type ATP synthase subunit F
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Soluble domain of V/A-ATPase including V1, d, two EG stalks, and ...

SupramoleculeName: Soluble domain of V/A-ATPase including V1, d, two EG stalks, and N-terminal of a-subunit
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 / Details: V/A-type ATPase from Thermus thermophilus
Source (natural)Organism: Thermus thermophilus HB8 (bacteria) / Strain: HB8
Molecular weightTheoretical: 500 KDa

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Macromolecule #1: V-type ATP synthase alpha chain

MacromoleculeName: V-type ATP synthase alpha chain / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO / EC number: H+-transporting two-sector ATPase
Source (natural)Organism: Thermus thermophilus HB8 (bacteria) / Strain: HB8
Molecular weightTheoretical: 63.69998 KDa
SequenceString: MIQGVIQKIA GPAVIAKGML GARMYDICKV GEEGLVGEII RLDGDTAFVQ VYEDTSGLKV GEPVVSTGLP LAVELGPGML NGIYDGIQR PLERIREKTG IYITRGVVVH ALDREKKWAW TPMVKPGDEV RGGMVLGTVP EFGFTHKILV PPDVRGRVKE V KPAGEYTV ...String:
MIQGVIQKIA GPAVIAKGML GARMYDICKV GEEGLVGEII RLDGDTAFVQ VYEDTSGLKV GEPVVSTGLP LAVELGPGML NGIYDGIQR PLERIREKTG IYITRGVVVH ALDREKKWAW TPMVKPGDEV RGGMVLGTVP EFGFTHKILV PPDVRGRVKE V KPAGEYTV EEPVVVLEDG TELKMYHTWP VRRARPVQRK LDPNTPFLTG MRILDVLFPV AMGGTAAIPG PFGSGKTVTQ QS LAKWSNA DVVVYVGCGE RGNEMTDVLV EFPELTDPKT GGPLMHRTVL IANTSNMPVA AREASIYVGV TIAEYFRDQG FSV ALMADS TSRWAEALRE ISSRLEEMPA EEGYPPYLAA RLAAFYERAG KVITLGGEEG AVTIVGAVSP PGGDMSEPVT QSTL RIVGA FWRLDASLAF RRHFPAINWN GSYSLFTSAL DPWYRENVAE DYPELRDAIS ELLQREAGLQ EIVQLVGPDA LQDAE RLVI EVGRIIREDF LQQNAYHEVD AYCSMKKAYG IMKMILAFYK EAEAAIKRGV SIDEILQLPV LERIGRARYV SEEEFP AYF EEAMKEIQGA FKALA

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Macromolecule #2: V-type ATP synthase beta chain

MacromoleculeName: V-type ATP synthase beta chain / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria) / Strain: HB8
Molecular weightTheoretical: 53.2195 KDa
SequenceString: MDLLKKEYTG ITYISGPLLF VENAKDLAYG AIVDIKDGTG RVRGGQVIEV SEEYAVIQVF EETTGLDLAT TSVSLVEDVA RLGVSKEML GRRFNGIGKP IDGLPPITPE KRLPITGLPL NPVARRKPEQ FIQTGISTID VMNTLVRGQK LPIFSGSGLP A NEIAAQIA ...String:
MDLLKKEYTG ITYISGPLLF VENAKDLAYG AIVDIKDGTG RVRGGQVIEV SEEYAVIQVF EETTGLDLAT TSVSLVEDVA RLGVSKEML GRRFNGIGKP IDGLPPITPE KRLPITGLPL NPVARRKPEQ FIQTGISTID VMNTLVRGQK LPIFSGSGLP A NEIAAQIA RQATVRPDLS GEGEKEEPFA VVFAAMGITQ RELSYFIQEF ERTGALSRSV LFLNKADDPT IERILTPRMA LT VAEYLAF EHDYHVLVIL TDMTNYCEAL REIGAAREEI PGRRGYPGYM YTDLATIYER AGVVEGKKGS VTQIPILSMP DDD RTHPIP DLTGYITEGQ IQLSRELHRK GIYPPIDPLP SLSRLMNNGV GKGKTREDHK QVSDQLYSAY ANGVDIRKLV AIIG EDALT ENDRRYLQFA DAFERFFINQ GQQNRSIEES LQIAWALLSM LPQGELKRIS KDHIGKYYGQ KLEEIWGAPQ ALD

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Macromolecule #3: V-type ATP synthase subunit D

MacromoleculeName: V-type ATP synthase subunit D / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria) / Strain: HB8
Molecular weightTheoretical: 24.715566 KDa
SequenceString: MSQVSPTRMN LLQRRGQLRL AQKGVDLLKK KRDALVAEFF GLVREAMEAR KALDQAAKEA YAALLLAQAF DGPEVVAGAA LGVPPLEGV EAEVENVWGS KVPRLKATFP DGALLSPVGT PAYTLEASRA FRRYAEALIR VANTETRLKK IGEEIKKTTR R VNALEQVV ...String:
MSQVSPTRMN LLQRRGQLRL AQKGVDLLKK KRDALVAEFF GLVREAMEAR KALDQAAKEA YAALLLAQAF DGPEVVAGAA LGVPPLEGV EAEVENVWGS KVPRLKATFP DGALLSPVGT PAYTLEASRA FRRYAEALIR VANTETRLKK IGEEIKKTTR R VNALEQVV IPGIRAQIRF IQQVLEQRER EDTFRLKRIK GKIEAREAEE EGGRPNPQVE IGAGL

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Macromolecule #4: V-type ATP synthase subunit F

MacromoleculeName: V-type ATP synthase subunit F / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria) / Strain: HB8
Molecular weightTheoretical: 11.294904 KDa
SequenceString:
MAVIADPETA QGFRLAGLEG YGASSAEEAQ SLLETLVERG GYALVAVDEA LLPDPERAVE RLMRGRDLPV LLPIAGLKEA FQGHDVEGY MRELVRKTIG FDIKL

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Macromolecule #5: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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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

Concentration3.0 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMTris-HClTris
150.0 mMNaClSodium chlorideSodium Chloride
GridModel: Quantifoil R1.2/1.3 / Material: MOLYBDENUM / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThe sample was purified from cell membrane of Thermus thermophilus and incorporated into nanodisc.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.5 µm / Calibrated defocus min: 2.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 75000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Frames/image: 1-34 / Number real images: 3694 / Average exposure time: 2.0 sec. / Average electron dose: 45.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 350000
Details: The particles selected from manually-selected 3084 micrographs.
CTF correctionSoftware - Name: Gctf
Startup modelType of model: OTHER / Details: Previous result of same enzyme.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION / Software - details: 3.0.7
Final 3D classificationNumber classes: 3 / Avg.num./class: 33 / Software - Name: RELION / Software - details: 3.0.7
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION / Software - details: 3.0.7
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Software - details: 3.0.7
Details: Focused classification on the soluble domain was carried out using RELION progarm.
Number images used: 71196
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5y5y

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation coefficient
Output model

PDB-6ly8:
V/A-ATPase from Thermus thermophilus, the soluble domain, including V1, d, two EG stalks, and N-terminal domain of a-subunit.

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