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- EMDB-42881: State 2 Yeast V-ATPase without Oxr1p bound -

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Basic information

Entry
Database: EMDB / ID: EMD-42881
TitleState 2 Yeast V-ATPase without Oxr1p bound
Map dataYeast V-ATPase in state 2
Sample
  • Complex: Yeast Vacuolar ATPase vitrified in presence of Oxr1p
KeywordsVacuolar ATPase / Proton pump / Oxr1p / MEMBRANE PROTEIN
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.1 Å
AuthorsKhan MM / Wilkens S
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM141908 United States
CitationJournal: EMBO Rep / Year: 2024
Title: Molecular mechanism of Oxr1p mediated disassembly of yeast V-ATPase.
Authors: Md Murad Khan / Stephan Wilkens /
Abstract: The eukaryotic vacuolar H-ATPase (V-ATPase) is regulated by reversible disassembly into autoinhibited V-ATPase and V proton channel subcomplexes. We recently reported that the TLDc protein Oxr1p ...The eukaryotic vacuolar H-ATPase (V-ATPase) is regulated by reversible disassembly into autoinhibited V-ATPase and V proton channel subcomplexes. We recently reported that the TLDc protein Oxr1p induces V-ATPase disassembly in vitro. Whether and how Oxr1p is involved in enzyme disassembly in vivo, however, is not known. Here, using yeast genetics and fluorescence microscopy, we show that Oxr1p is essential for efficient V-ATPase disassembly in the cell. Supporting biochemical and biophysical in vitro experiments show that whereas Oxr1p-driven holoenzyme disassembly can occur in the absence of nucleotides, the presence of ATP greatly accelerates the process. ATP hydrolysis is needed, however, for subsequent release of Oxr1p so that the free V can adopt the autoinhibited conformation. Overall, our study unravels the molecular mechanism of Oxr1p-induced disassembly that occurs in vivo as part of the canonical V-ATPase regulation by reversible disassembly.
History
DepositionNov 19, 2023-
Header (metadata) releaseApr 10, 2024-
Map releaseApr 10, 2024-
UpdateApr 10, 2024-
Current statusApr 10, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42881.png

Downloads & links

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Map

FileDownload / File: emd_42881.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationYeast V-ATPase in state 2
Voxel sizeX=Y=Z: 2.85 Å
Density
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.025723813 - 0.087895066
Average (Standard dev.)0.00020915495 (±0.005338186)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 456.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_42881_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_42881_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_42881_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Yeast Vacuolar ATPase vitrified in presence of Oxr1p

EntireName: Yeast Vacuolar ATPase vitrified in presence of Oxr1p
Components
  • Complex: Yeast Vacuolar ATPase vitrified in presence of Oxr1p

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Supramolecule #1: Yeast Vacuolar ATPase vitrified in presence of Oxr1p

SupramoleculeName: Yeast Vacuolar ATPase vitrified in presence of Oxr1p / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Organelle: Vacuole
Molecular weightTheoretical: 1 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2 mg/mL
BufferpH: 7.4 / Details: TBS
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 279 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeJEOL 2100F
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.2 µm
Sample stageSpecimen holder model: GATAN 914 HIGH TILT LIQUID NITROGEN CRYO TRANSFER TOMOGRAPHY HOLDER
Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 50.0 e/Å2

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

31538

Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 4)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4) / Number images used: 13950
FSC plot (resolution estimation)

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