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- PDB-6ly9: The membrane-embedded Vo domain of V/A-ATPase from Thermus thermo... -

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Basic information

Entry
Database: PDB / ID: 6ly9
TitleThe membrane-embedded Vo domain of V/A-ATPase from Thermus thermophilus
Components
  • V-type ATP synthase subunit C
  • V-type ATP synthase subunit E
  • V-type ATP synthase subunit I
  • V-type ATP synthase, subunit (VAPC-THERM)
  • V-type ATP synthase, subunit K
KeywordsMOTOR PROTEIN / rotary ATPase / V/A-ATPase / molecular motor
Function / homology
Function and homology information


proton-transporting V-type ATPase, V0 domain / proton-transporting two-sector ATPase complex, catalytic domain / proton motive force-driven plasma membrane ATP synthesis / proton-transporting ATPase activity, rotational mechanism / proton-transporting ATP synthase activity, rotational mechanism / ATP binding / metal ion binding
Similarity search - Function
V-type ATP synthase subunit I, N-terminal / Vacuolar ATPase subunit I, N-terminal proximal lobe / Vacuolar ATPase Subunit I N-terminal proximal lobe / V-type ATPase subunit I, N-terminal domain / ATPase, V0 complex, c subunit / Vacuolar (H+)-ATPase G subunit / V-ATPase proteolipid subunit / ATPase, V0 complex, c/d subunit / V-type ATPase subunit C/d / V-type ATP synthase subunit c/d subunit superfamily ...V-type ATP synthase subunit I, N-terminal / Vacuolar ATPase subunit I, N-terminal proximal lobe / Vacuolar ATPase Subunit I N-terminal proximal lobe / V-type ATPase subunit I, N-terminal domain / ATPase, V0 complex, c subunit / Vacuolar (H+)-ATPase G subunit / V-ATPase proteolipid subunit / ATPase, V0 complex, c/d subunit / V-type ATPase subunit C/d / V-type ATP synthase subunit c/d subunit superfamily / V-type ATP synthase c/d subunit, domain 3 superfamily / ATP synthase (C/AC39) subunit / V-type ATPase, V0 complex, 116kDa subunit family / V-type ATPase 116kDa subunit family / V-type ATPase subunit E / V-type ATPase subunit E, C-terminal domain superfamily / ATP synthase (E/31 kDa) subunit / V-ATPase proteolipid subunit C-like domain / F/V-ATP synthase subunit C superfamily / ATP synthase subunit C
Similarity search - Domain/homology
V-type ATP synthase subunit E / V-type ATP synthase subunit C / V-type ATP synthase, subunit (VAPC-THERM) / V-type ATP synthase subunit I / V-type ATP synthase, subunit K
Similarity search - Component
Biological speciesThermus thermophilus HB8 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.93 Å
AuthorsKishikawa, J. / Nakanishi, A. / Furuta, A. / Kato, T. / Namba, K. / Tamakoshi, M. / Mitsuoka, K. / Yokoyama, K.
Funding support Japan, 3items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)17H03648 Japan
Japan Agency for Medical Research and Development (AMED)JP17am0101001 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)12024046 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, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 9, 2020Provider: repository / Type: Initial release
Revision 1.1Sep 30, 2020Group: Database references / Category: citation / Item: _citation.title

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Structure visualization

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Assembly

Deposited unit
N: V-type ATP synthase subunit I
O: V-type ATP synthase, subunit K
P: V-type ATP synthase, subunit K
Q: V-type ATP synthase, subunit K
R: V-type ATP synthase, subunit K
S: V-type ATP synthase, subunit K
T: V-type ATP synthase, subunit K
U: V-type ATP synthase, subunit K
V: V-type ATP synthase, subunit K
W: V-type ATP synthase, subunit K
X: V-type ATP synthase, subunit K
Y: V-type ATP synthase, subunit K
Z: V-type ATP synthase, subunit K
M: V-type ATP synthase subunit C
K: V-type ATP synthase, subunit (VAPC-THERM)
L: V-type ATP synthase subunit E


Theoretical massNumber of molelcules
Total (without water)260,08516
Polymers260,08516
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis, The subunits co-migrate in Native-PAGE
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein V-type ATP synthase subunit I


Mass: 72204.289 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / Strain: HB8 / References: UniProt: Q5SIT6
#2: Protein
V-type ATP synthase, subunit K


Mass: 9841.714 Da / Num. of mol.: 12 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / Strain: HB8 / References: UniProt: Q5SIT7
#3: Protein V-type ATP synthase subunit C / V-ATPase subunit C


Mass: 35968.570 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / Strain: HB8 / References: UniProt: P74902
#4: Protein V-type ATP synthase, subunit (VAPC-THERM)


Mass: 13166.218 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / Strain: HB8 / References: UniProt: Q5SIT5
#5: Protein V-type ATP synthase subunit E / V-ATPase subunit E


Mass: 20645.582 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / Strain: HB8 / References: UniProt: P74901

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Membrane-embedded Vo domain / Type: COMPLEX / Details: V/A-type ATPase from Thermus thermophilus / Entity ID: all / Source: NATURAL
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.650 MDaNO
210.26 MDaNO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris-HClTris1
2150 mMSodium ChlorideNaClSodium chloride1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample was purified from cell membrane of Thermus thermophilus and incorporated into nanodisc.
Specimen supportGrid material: MOLYBDENUM / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 200
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000 X / Calibrated defocus min: 1000 nm / Calibrated defocus max: 2000 nm / Cs: 1.4 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 12 sec. / Electron dose: 79.2 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 5988
EM imaging opticsEnergyfilter name: In-column Omega Filter
Image scansWidth: 4096 / Height: 4096 / Movie frames/image: 60 / Used frames/image: 1-60
EM diffractionCamera length: 800 mm

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Processing

SoftwareName: PHENIX / Classification: refinement
EM software
IDNameCategoryDetails
4GctfCTF correction
7Cootmodel fitting
9RELIONinitial Euler assignment3.0.7
10RELIONfinal Euler assignment3.0.7
11RELIONclassification3.0.7
12RELION3D reconstruction3.0.7
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3140000
Details: The particles selected from manually-selected 3268 micrographs.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.93 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 157618 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 6QUM

6qum

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00614105
ELECTRON MICROSCOPYf_angle_d0.7119085
ELECTRON MICROSCOPYf_dihedral_angle_d13.1018205
ELECTRON MICROSCOPYf_chiral_restr0.0432254
ELECTRON MICROSCOPYf_plane_restr0.0042456

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