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- PDB-6ly8: V/A-ATPase from Thermus thermophilus, the soluble domain, includi... -

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Basic information

Entry
Database: PDB / ID: 6ly8
TitleV/A-ATPase from Thermus thermophilus, the soluble domain, including V1, d, two EG stalks, and N-terminal domain of a-subunit.
Components
  • V-type ATP synthase alpha chain
  • V-type ATP synthase beta chain
  • V-type ATP synthase subunit D
  • V-type ATP synthase subunit F
KeywordsMOTOR PROTEIN / rotary ATPase / V/A-ATPase / molecular motor
Function / homology
Function and homology information


plant-type vacuole / vacuolar proton-transporting V-type ATPase complex / proton-transporting ATP synthase complex / vacuolar acidification / fungal-type vacuole membrane / proton motive force-driven plasma membrane ATP synthesis / H+-transporting two-sector ATPase / phagocytic vesicle / proton-transporting ATPase activity, rotational mechanism / proton-transporting ATP synthase activity, rotational mechanism ...plant-type vacuole / vacuolar proton-transporting V-type ATPase complex / proton-transporting ATP synthase complex / vacuolar acidification / fungal-type vacuole membrane / proton motive force-driven plasma membrane ATP synthesis / H+-transporting two-sector ATPase / phagocytic vesicle / proton-transporting ATPase activity, rotational mechanism / proton-transporting ATP synthase activity, rotational mechanism / lysosomal membrane / 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
ADENOSINE-5'-DIPHOSPHATE / 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)
MethodELECTRON MICROSCOPY / single 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, 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
A: V-type ATP synthase alpha chain
B: V-type ATP synthase alpha chain
C: V-type ATP synthase alpha chain
D: V-type ATP synthase beta chain
E: V-type ATP synthase beta chain
F: V-type ATP synthase beta chain
G: V-type ATP synthase subunit D
H: V-type ATP synthase subunit F
hetero molecules


Theoretical massNumber of molelcules
Total (without water)387,1969
Polymers386,7698
Non-polymers4271
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis, The subunits are co-migrate in Native-PAGE.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area34230 Å2
ΔGint-145 kcal/mol
Surface area132100 Å2

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Components

#1: Protein V-type ATP synthase alpha chain / V-ATPase subunit A


Mass: 63699.980 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / Strain: HB8
References: UniProt: Q56403, H+-transporting two-sector ATPase
#2: Protein V-type ATP synthase beta chain / V-ATPase subunit B


Mass: 53219.500 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / Strain: HB8 / References: UniProt: Q56404
#3: Protein V-type ATP synthase subunit D / V-ATPase subunit D


Mass: 24715.566 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / Strain: HB8 / References: UniProt: O87880
#4: Protein V-type ATP synthase subunit F / V-ATPase subunit F


Mass: 11294.904 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / Strain: HB8 / References: UniProt: P74903
#5: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestN

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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: Soluble domain of V/A-ATPase including V1, d, two EG stalks, and N-terminal of a-subunit
Type: COMPLEX / Details: V/A-type ATPase from Thermus thermophilus / Entity ID: #1-#4 / Source: NATURAL
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
11650 kDa/nmNO
210.5 MDaNO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria) / Strain: HB8
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris-HCl1
2150 mMSodium ChlorideNaCl1
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

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 75000 X / Calibrated defocus min: 2000 nm / Calibrated defocus max: 3500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 2 sec. / Electron dose: 45 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of real images: 3694
Image scansWidth: 4096 / Height: 4096 / Movie frames/image: 34 / Used frames/image: 1-34
EM diffractionCamera length: 800 mm

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Processing

SoftwareName: PHENIX / Classification: refinement
EM software
IDNameCategoryDetails
2EPUimage acquisition
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: 350000
Details: The particles selected from manually-selected 3084 micrographs.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 71196 / Algorithm: FOURIER SPACE
Details: Focused classification on the soluble domain was carried out using RELION progarm.
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 5Y5Y

5y5y

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00627153
ELECTRON MICROSCOPYf_angle_d0.6536793
ELECTRON MICROSCOPYf_dihedral_angle_d13.3816494
ELECTRON MICROSCOPYf_chiral_restr0.0464109
ELECTRON MICROSCOPYf_plane_restr0.0054818

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