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- PDB-7fde: CryoEM Structures of Reconstituted V-ATPase, Oxr1 bound V1 -

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Basic information

Entry
Database: PDB / ID: 7fde
TitleCryoEM Structures of Reconstituted V-ATPase, Oxr1 bound V1
Components
  • (V-type proton ATPase subunit ...) x 6
  • Oxidation resistance protein 1
  • Yeast Vacuolar ATPase A subunit
KeywordsMOTOR PROTEIN / ATPase / proton pump / rotary motor enzyme / membrane protein
Function / homology
Function and homology information


vacuole-mitochondrion membrane contact site / Insulin receptor recycling / Transferrin endocytosis and recycling / ROS and RNS production in phagocytes / Amino acids regulate mTORC1 / Golgi lumen acidification / proteasome storage granule assembly / vacuolar proton-transporting V-type ATPase, V1 domain / endosomal lumen acidification / pexophagy ...vacuole-mitochondrion membrane contact site / Insulin receptor recycling / Transferrin endocytosis and recycling / ROS and RNS production in phagocytes / Amino acids regulate mTORC1 / Golgi lumen acidification / proteasome storage granule assembly / vacuolar proton-transporting V-type ATPase, V1 domain / endosomal lumen acidification / pexophagy / proton-transporting V-type ATPase complex / vacuolar proton-transporting V-type ATPase complex / vacuolar acidification / fungal-type vacuole membrane / ATP metabolic process / Neutrophil degranulation / proton transmembrane transport / proton-transporting ATPase activity, rotational mechanism / transmembrane transport / intracellular calcium ion homeostasis / cytoplasmic stress granule / cellular response to oxidative stress / response to oxidative stress / Golgi membrane / mitochondrion / ATP binding / membrane / nucleus / cytoplasm
Similarity search - Function
TLDc domain profile. / TLDc domain / TLD / domain in TBC and LysM domain containing proteins / 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 / V-type ATPase subunit E ...TLDc domain profile. / TLDc domain / TLD / domain in TBC and LysM domain containing proteins / 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 / 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 / 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 proton ATPase subunit B / V-type proton ATPase subunit E / V-type proton ATPase subunit C / V-type proton ATPase subunit D / V-type proton ATPase subunit F / Oxidation resistance protein 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 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 29, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
O: V-type proton ATPase subunit C
G: V-type proton ATPase subunit E
H: V-type proton ATPase subunit G
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
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
I: V-type proton ATPase subunit E
J: V-type proton ATPase subunit G
P: Oxidation resistance protein 1


Theoretical massNumber of molelcules
Total (without water)615,34116
Polymers615,34116
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area66660 Å2
ΔGint-381 kcal/mol
Surface area213880 Å2

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Components

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V-type proton ATPase subunit ... , 6 types, 12 molecules OGKIHLJBDFMN

#1: 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 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta2 / References: UniProt: P31412
#2: 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
#3: 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 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
#6: 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
#7: 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

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Protein , 2 types, 4 molecules ACEP

#4: 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
#8: Protein Oxidation resistance protein 1


Mass: 30818.334 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / References: UniProt: Q08952

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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
1Oxr1 bound V1 subcomplex of Yeast Vacuolar ATPaseCOMPLEXall0MULTIPLE SOURCES
2Yeast Vacuolar ATPase subunit CV-ATPaseCOMPLEX#11RECOMBINANT
3Yeast V-type proton ATPase subunitsCOMPLEX#2-#81NATURALV-ATPase subunits purified from natural source
Molecular weightValue: 0.6 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Saccharomyces cerevisiae S288c (yeast)559292
22Saccharomyces cerevisiae S288c (yeast)559292
Source (recombinant)Organism: Escherichia coli (E. coli)
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 FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
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: 20447
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: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 20447 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00240254
ELECTRON MICROSCOPYf_angle_d0.48554387
ELECTRON MICROSCOPYf_dihedral_angle_d4.4645466
ELECTRON MICROSCOPYf_chiral_restr0.0396136
ELECTRON MICROSCOPYf_plane_restr0.0037063

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