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- PDB-7fdb: CryoEM Structures of Reconstituted V-ATPase,State2 -

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Basic information

Entry
Database: PDB / ID: 7fdb
TitleCryoEM Structures of Reconstituted V-ATPase,State2
Components
  • (V-type proton ATPase subunit ...) x 11
  • (Yeast Vacuolar ATPase ...) x 3
  • Fusion of yeast V-type proton ATPase subunit H(NT) and human V-type proton ATPase subunit H(CT)
  • V0 assembly protein 1
KeywordsMOTOR PROTEIN / ATPase / proton pump / rotary motor enzyme / membrane protein
Function / homology
Function and homology information


Blockage of phagosome acidification / Ion channel transport / Regulation of MITF-M-dependent genes involved in lysosome biogenesis and autophagy / intracellular pH reduction / vacuole-mitochondrion membrane contact site / cell wall mannoprotein biosynthetic process / Nef Mediated CD8 Down-regulation / ATPase-coupled ion transmembrane transporter activity / protein localization to vacuolar membrane / cellular response to alkaline pH ...Blockage of phagosome acidification / Ion channel transport / Regulation of MITF-M-dependent genes involved in lysosome biogenesis and autophagy / intracellular pH reduction / vacuole-mitochondrion membrane contact site / cell wall mannoprotein biosynthetic process / Nef Mediated CD8 Down-regulation / ATPase-coupled ion transmembrane transporter activity / protein localization to vacuolar membrane / cellular response to alkaline pH / Insulin receptor recycling / Transferrin endocytosis and recycling / polyphosphate metabolic process / ROS and RNS production in phagocytes / Amino acids regulate mTORC1 / Golgi lumen acidification / proteasome storage granule assembly / synaptic vesicle lumen acidification / Transferrin endocytosis and recycling / extrinsic component of synaptic vesicle membrane / P-type proton-exporting transporter activity / lysosomal lumen acidification / clathrin-coated vesicle membrane / pexophagy / vacuolar transport / endosomal lumen acidification / vacuolar proton-transporting V-type ATPase, V0 domain / vacuolar proton-transporting V-type ATPase, V1 domain / protein targeting to vacuole / vacuole organization / vacuolar proton-transporting V-type ATPase complex / Amino acids regulate mTORC1 / proton-transporting V-type ATPase complex / fungal-type vacuole / vacuolar acidification / ROS and RNS production in phagocytes / cellular hyperosmotic response / Nef Mediated CD4 Down-regulation / fungal-type vacuole membrane / phosphatidylinositol-3,5-bisphosphate binding / proton transmembrane transporter activity / intracellular copper ion homeostasis / regulation of macroautophagy / enzyme regulator activity / ATP metabolic process / Insulin receptor recycling / Neutrophil degranulation / proton-transporting ATPase activity, rotational mechanism / RNA endonuclease activity / proton transmembrane transport / cell periphery / transmembrane transport / intracellular calcium ion homeostasis / cytoplasmic stress granule / endocytosis / ATPase binding / protein-containing complex assembly / intracellular iron ion homeostasis / endosome membrane / membrane raft / lysosomal membrane / Golgi membrane / endoplasmic reticulum membrane / extracellular exosome / ATP binding / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
ATPase, V1 complex, subunit H / ATPase, V1 complex, subunit H, C-terminal / ATPase, V1 complex, subunit H, C-terminal domain superfamily / V-ATPase subunit H / V-ATPase subunit H / Ribonuclease kappa / V-type proton ATPase subunit S1/VOA1, transmembrane domain / V0 complex accessory subunit Ac45/VOA1 transmembrane domain / ATPase, V1 complex, subunit C / Vacuolar ATP synthase subunit C superfamily ...ATPase, V1 complex, subunit H / ATPase, V1 complex, subunit H, C-terminal / ATPase, V1 complex, subunit H, C-terminal domain superfamily / V-ATPase subunit H / V-ATPase subunit H / Ribonuclease kappa / V-type proton ATPase subunit S1/VOA1, transmembrane domain / V0 complex accessory subunit Ac45/VOA1 transmembrane domain / ATPase, V1 complex, subunit C / Vacuolar ATP synthase subunit C superfamily / V-ATPase subunit C / ATPase, V1 complex, subunit B / ATPase, V1 complex, subunit F, eukaryotic / ATPase, V0 complex, subunit e1/e2 / ATP synthase subunit H / ATPase, V0 complex, subunit d / V-ATPase proteolipid subunit C, eukaryotic / ATPase, V0 complex, subunit 116kDa, eukaryotic / 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-ATPase proteolipid 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 / 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-ATPase proteolipid subunit C-like domain / F/V-ATP synthase subunit C superfamily / ATP synthase subunit C / C-terminal domain of V and A type ATP synthase / 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 / Armadillo-like helical / Armadillo-type fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
V-type proton ATPase subunit f / V-type proton ATPase subunit B / V-type proton ATPase subunit E / V-type proton ATPase subunit c'' / V-type proton ATPase subunit c / V-type proton ATPase subunit C / V-type proton ATPase subunit d / V-type proton ATPase subunit a, vacuolar isoform / V-type proton ATPase subunit D / V-type proton ATPase subunit c' ...V-type proton ATPase subunit f / V-type proton ATPase subunit B / V-type proton ATPase subunit E / V-type proton ATPase subunit c'' / V-type proton ATPase subunit c / V-type proton ATPase subunit C / V-type proton ATPase subunit d / V-type proton ATPase subunit a, vacuolar isoform / V-type proton ATPase subunit D / V-type proton ATPase subunit c' / V-type proton ATPase subunit F / V-type proton ATPase subunit H / V0 assembly protein 1 / V-type proton ATPase subunit e / V-type proton ATPase subunit H
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsKhan, M.M. / Lee, S. / Oot, R.A. / Couoh-Cardel, S. / KIm, H. / Wilkens, S. / Roh, S.H.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM058600 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA228340 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM141908 United States
CitationJournal: EMBO J / Year: 2022
Title: Oxidative stress protein Oxr1 promotes V-ATPase holoenzyme disassembly in catalytic activity-independent manner.
Authors: Md Murad Khan / Seowon Lee / Sergio Couoh-Cardel / Rebecca A Oot / Hyunmin Kim / Stephan Wilkens / Soung-Hun Roh /
Abstract: The vacuolar ATPase (V-ATPase) is a rotary motor proton pump that is regulated by an assembly equilibrium between active holoenzyme and autoinhibited V -ATPase and V proton channel subcomplexes. ...The vacuolar ATPase (V-ATPase) is a rotary motor proton pump that is regulated by an assembly equilibrium between active holoenzyme and autoinhibited V -ATPase and V proton channel subcomplexes. Here, we report cryo-EM structures of yeast V-ATPase assembled in vitro from lipid nanodisc reconstituted V and mutant V . Our analysis identified holoenzymes in three active rotary states, indicating that binding of V to V provides sufficient free energy to overcome V autoinhibition. Moreover, the structures suggest that the unequal spacing of V 's proton-carrying glutamic acid residues serves to alleviate the symmetry mismatch between V and V motors, a notion that is supported by mutagenesis experiments. We also uncover a structure of free V bound to Oxr1, a conserved but poorly characterized factor involved in the oxidative stress response. Biochemical experiments show that Oxr1 inhibits V -ATPase and causes disassembly of the holoenzyme, suggesting that Oxr1 plays a direct role in V-ATPase regulation.
History
DepositionJul 16, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 22, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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Assembly

Deposited unit
A: Yeast Vacuolar ATPase A subunit
B: V-type proton ATPase subunit B
C: Yeast Vacuolar ATPase A subunit
D: V-type proton ATPase subunit B
E: Yeast Vacuolar ATPase A subunit
F: V-type proton ATPase subunit B
G: V-type proton ATPase subunit E
H: V-type proton ATPase subunit G
I: V-type proton ATPase subunit E
J: V-type proton ATPase subunit G
K: V-type proton ATPase subunit E
L: V-type proton ATPase subunit G
M: V-type proton ATPase subunit D
N: V-type proton ATPase subunit F
O: V-type proton ATPase subunit C
P: Fusion of yeast V-type proton ATPase subunit H(NT) and human V-type proton ATPase subunit H(CT)
Q: Yeast Vacuolar ATPase a subunit
S: V-type proton ATPase subunit d
T: V-type proton ATPase subunit c''
U: V-type proton ATPase subunit c'
V: V-type proton ATPase subunit c
W: V-type proton ATPase subunit c
X: V-type proton ATPase subunit c
Y: V-type proton ATPase subunit c
Z: V-type proton ATPase subunit c
a: V-type proton ATPase subunit c
b: V-type proton ATPase subunit c
c: V-type proton ATPase subunit c
d: V-type proton ATPase subunit e
e: V0 assembly protein 1
f: Yeast Vacuolar ATPase f subunit


Theoretical massNumber of molelcules
Total (without water)991,82531
Polymers991,82531
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, Concanamycin A sensitive MgATPase activity
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area121460 Å2
ΔGint-912 kcal/mol
Surface area354040 Å2

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Components

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Yeast Vacuolar ATPase ... , 3 types, 5 molecules ACEQf

#1: Protein Yeast Vacuolar ATPase A subunit


Mass: 67796.508 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / References: H+-transporting two-sector ATPase
#9: Protein Yeast Vacuolar ATPase a subunit / V-ATPase a 1 subunit


Mass: 95625.484 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / References: UniProt: P32563
#16: Protein Yeast Vacuolar ATPase f subunit


Mass: 9369.934 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / References: UniProt: P0C5R9

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V-type proton ATPase subunit ... , 11 types, 24 molecules BDFGIKHJLMNOSTUVWXYZabcd

#2: Protein V-type proton ATPase subunit B / V-ATPase subunit B


Mass: 57815.023 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / References: UniProt: P16140
#3: Protein V-type proton ATPase subunit E / V-ATPase subunit E


Mass: 26508.393 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / References: UniProt: P22203
#4: Protein V-type proton ATPase subunit G


Mass: 13735.680 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c
#5: Protein V-type proton ATPase subunit D / V-ATPase subunit D


Mass: 29235.023 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / References: UniProt: P32610
#6: Protein V-type proton ATPase subunit F / V-ATPase subunit F


Mass: 13479.170 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / References: UniProt: P39111
#7: Protein V-type proton ATPase subunit C / V-ATPase subunit C


Mass: 44241.352 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / Gene: VMA5 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta2 / References: UniProt: P31412
#10: Protein V-type proton ATPase subunit d / V-ATPase subunit d


Mass: 39822.484 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / References: UniProt: P32366
#11: Protein V-type proton ATPase subunit c'' / V-ATPase subunit c''


Mass: 22610.641 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / References: UniProt: P23968
#12: Protein V-type proton ATPase subunit c' / V-ATPase subunit c'


Mass: 17046.361 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / References: UniProt: P32842
#13: Protein
V-type proton ATPase subunit c / V-ATPase subunit c


Mass: 16357.501 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / References: UniProt: P25515
#14: Protein V-type proton ATPase subunit e / V-ATPase subunit e


Mass: 8387.065 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / References: UniProt: Q3E7B6

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Protein , 2 types, 2 molecules Pe

#8: Protein Fusion of yeast V-type proton ATPase subunit H(NT) and human V-type proton ATPase subunit H(CT) / V-ATPase subunit H


Mass: 53885.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast), (gene. exp.) Homo sapiens (human)
Strain: S288c / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta2 / References: UniProt: P41807, UniProt: Q9UI12
#15: Protein V0 assembly protein 1


Mass: 29694.885 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / References: UniProt: P53262

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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

Component
IDNameTypeEntity IDParent-IDSourceDetails
1Yeast Vacuolar ATPase in rotary state 2COMPLEXall0MULTIPLE SOURCES
2Yeast Vacuolar ATPase C subunitCOMPLEX#71RECOMBINANT
3Chimeric subunit HCOMPLEX#81RECOMBINANTFusion of yeast V-type proton ATPase subunit H(NT) and human V-type proton ATPase subunit H(CT)
4V-type proton ATPase subunitsCOMPLEX#2-#6, #9-#161NATURALV-ATPase subunits purified from natural source
Molecular weightValue: 1 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Saccharomyces cerevisiae S288c (yeast)559292
22Saccharomyces cerevisiae S288c (yeast)559292
32Homo sapiens (human)9606
43Saccharomyces cerevisiae S288c (yeast)559292
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
11Escherichia coli (E. coli)562
22Escherichia coli (E. coli)562
32Escherichia coli (E. coli)562
Buffer solutionpH: 7.4
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingAverage exposure time: 10 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 42206
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.19rc6_4061: / Classification: refinement
EM software
IDNameCategory
2EPUimage acquisition
7Cootmodel fitting
9PHENIXmodel refinement
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 42206 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00365666
ELECTRON MICROSCOPYf_angle_d0.53388844
ELECTRON MICROSCOPYf_dihedral_angle_d4.8228920
ELECTRON MICROSCOPYf_chiral_restr0.03810243
ELECTRON MICROSCOPYf_plane_restr0.00411334

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