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- PDB-8gxx: 3 nucleotide-bound V1EG of V/A-ATPase from Thermus thermophilus. -

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Basic information

Entry
Database: PDB / ID: 8gxx
Title3 nucleotide-bound V1EG of V/A-ATPase from Thermus thermophilus.
Components
  • (V-type ATP synthase ...) x 5
  • V-type ATP synthase, subunit (VAPC-THERM)
KeywordsHYDROLASE / rotary ATPase / V/A-type ATPase / V-ATPase
Function / homology
Function and homology information


proton-transporting two-sector ATPase complex, catalytic domain / proton-transporting V-type ATPase complex / proton motive force-driven plasma membrane ATP synthesis / proton-transporting ATP synthase complex / H+-transporting two-sector ATPase / proton-transporting ATPase activity, rotational mechanism / proton-transporting ATP synthase activity, rotational mechanism / ATP binding / metal ion binding
Similarity search - Function
Vacuolar (H+)-ATPase G subunit / ATPase, V1 complex, subunit F, bacterial/archaeal / ATPase, V1 complex, subunit D / V-type ATPase subunit E / ATPase, V1 complex, subunit F / ATPase, V1 complex, subunit F superfamily / V-type ATPase subunit E, C-terminal domain superfamily / ATP synthase subunit D / ATP synthase (F/14-kDa) subunit / ATP synthase (E/31 kDa) subunit ...Vacuolar (H+)-ATPase G subunit / ATPase, V1 complex, subunit F, bacterial/archaeal / ATPase, V1 complex, subunit D / V-type ATPase subunit E / ATPase, V1 complex, subunit F / ATPase, V1 complex, subunit F superfamily / V-type ATPase subunit E, C-terminal domain superfamily / ATP synthase subunit D / ATP synthase (F/14-kDa) subunit / ATP synthase (E/31 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 barrel-sandwich domain / : / ATPase, F1/V1 complex, beta/alpha subunit, C-terminal / C-terminal domain of V and A type ATP synthase / 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 / ADENOSINE-5'-TRIPHOSPHATE / V-type ATP synthase subunit D / V-type ATP synthase subunit E / V-type ATP synthase subunit F / V-type ATP synthase alpha chain / V-type ATP synthase beta chain / V-type ATP synthase, subunit (VAPC-THERM)
Similarity search - Component
Biological speciesThermus thermophilus HB8 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsNakanishi, A. / Kishikawa, J. / Mitsuoka, K. / Yokoyama, K.
Funding support Japan, 5items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)20H03231 Japan
Japan Society for the Promotion of Science (JSPS)20J00162 Japan
Japan Society for the Promotion of Science (JSPS)20K06514 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)JPMXP09A21OS0008 Japan
Japan Agency for Medical Research and Development (AMED)JP17am0101001 Japan
CitationJournal: J Biol Chem / Year: 2023
Title: Cryo-EM analysis of V/A-ATPase intermediates reveals the transition of the ground-state structure to steady-state structures by sequential ATP binding.
Authors: Atsuko Nakanishi / Jun-Ichi Kishikawa / Kaoru Mitsuoka / Ken Yokoyama /
Abstract: Vacuolar/archaeal-type ATPase (V/A-ATPase) is a rotary ATPase that shares a common rotary catalytic mechanism with FF ATP synthase. Structural images of V/A-ATPase obtained by single-particle cryo- ...Vacuolar/archaeal-type ATPase (V/A-ATPase) is a rotary ATPase that shares a common rotary catalytic mechanism with FF ATP synthase. Structural images of V/A-ATPase obtained by single-particle cryo-electron microscopy during ATP hydrolysis identified several intermediates, revealing the rotary mechanism under steady-state conditions. However, further characterization is needed to understand the transition from the ground state to the steady state. Here, we identified the cryo-electron microscopy structures of V/A-ATPase corresponding to short-lived initial intermediates during the activation of the ground state structure by time-resolving snapshot analysis. These intermediate structures provide insights into how the ground-state structure changes to the active, steady state through the sequential binding of ATP to its three catalytic sites. All the intermediate structures of V/A-ATPase adopt the same asymmetric structure, whereas the three catalytic dimers adopt different conformations. This is significantly different from the initial activation process of FF, where the overall structure of the F domain changes during the transition from a pseudo-symmetric to a canonical asymmetric structure (PNAS NEXUS, pgac116, 2022). In conclusion, our findings provide dynamical information that will enhance the future prospects for studying the initial activation processes of the enzymes, which have unknown intermediate structures in their functional pathway.
History
DepositionSep 21, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 25, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 1, 2023Group: Database references / Category: citation
Item: _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Feb 15, 2023Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.3Jul 3, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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
I: V-type ATP synthase, subunit (VAPC-THERM)
J: V-type ATP synthase subunit E
K: V-type ATP synthase, subunit (VAPC-THERM)
L: V-type ATP synthase subunit E
hetero molecules


Theoretical massNumber of molelcules
Total (without water)455,79317
Polymers454,30212
Non-polymers1,4905
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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V-type ATP synthase ... , 5 types, 10 molecules ABCDEFGHJL

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


Mass: 63669.957 Da / Num. of mol.: 3 / Source method: isolated from a natural source
Details: Authors state that the bacterium they used has two mutations in its genome (S232A and T235S) and they obtained the EM sample from 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
#6: Protein V-type ATP synthase subunit E / V-ATPase subunit E


Mass: 20645.582 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / Strain: HB8 / References: UniProt: P74901

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Protein , 1 types, 2 molecules IK

#5: Protein V-type ATP synthase, subunit (VAPC-THERM)


Mass: 13166.218 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / Strain: HB8 / References: UniProt: Q5SIT5

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Non-polymers , 3 types, 5 molecules

#7: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM

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Details

Has ligand of interestY

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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: 3 nucleotide-bound V1EG of V/A-ATPase from Thermus thermophilus.
Type: COMPLEX / Entity ID: #1-#6 / Source: NATURAL
Molecular weightValue: 0.6 MDa / Experimental value: NO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 defocus max: 1800 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20rc4_4425: / Classification: refinement
EM software
IDNameCategory
1RELIONparticle selection
2SerialEMimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 19234 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 7VAL

7val


Accession code: 7VAL / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00630110
ELECTRON MICROSCOPYf_angle_d0.54240807
ELECTRON MICROSCOPYf_dihedral_angle_d11.4811403
ELECTRON MICROSCOPYf_chiral_restr0.0444572
ELECTRON MICROSCOPYf_plane_restr0.0055333

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