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- EMDB-5476: Structure of the vacuolar-type ATPase from Saccharomyces cerevisi... -

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Basic information

Entry
Database: EMDB / ID: EMD-5476
TitleStructure of the vacuolar-type ATPase from Saccharomyces cerevisiae at 11 Angstrom resolution
Map data3D map of V-type ATPase
Sample
  • Sample: vacuolar-type ATPases
  • Protein or peptide: vacuolar-type ATPases
Keywordsmembrane protein / proton pump / ATPase / vacuole / endosome / lysosome / plasma membrane / Golgi
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 11.0 Å
AuthorsBenlekbir S / Bueler SA / Rubinstein JL
CitationJournal: Nat Struct Mol Biol / Year: 2012
Title: Structure of the vacuolar-type ATPase from Saccharomyces cerevisiae at 11-Å resolution.
Authors: Samir Benlekbir / Stephanie A Bueler / John L Rubinstein /
Abstract: Vacuolar-type ATPases (V-type ATPases) in eukaryotic cells are large membrane protein complexes that acidify various intracellular compartments. The enzymes are regulated by dissociation of the V(1) ...Vacuolar-type ATPases (V-type ATPases) in eukaryotic cells are large membrane protein complexes that acidify various intracellular compartments. The enzymes are regulated by dissociation of the V(1) and V(O) regions of the complex. Here we present the structure of the Saccharomyces cerevisiae V-type ATPase at 11-Å resolution by cryo-EM of protein particles in ice. The structure explains many cross-linking and protein interaction studies. Docking of crystal structures suggests that inhibition of ATPase activity by the dissociated V(1) region involves rearrangement of the N- and C-terminal domains of subunit H and also suggests how this inhibition is triggered upon dissociation. We provide support for this model by demonstrating that mutation of subunit H to increase the rigidity of the linker between its two domains decreases its ability to inhibit ATPase activity.
History
DepositionAug 22, 2012-
Header (metadata) releaseNov 7, 2012-
Map releaseNov 7, 2012-
UpdateDec 19, 2012-
Current statusDec 19, 2012Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.76
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.76
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5476_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5476.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D map of V-type ATPase
Voxel sizeX=Y=Z: 1.4 Å
Density
Contour LevelBy AUTHOR: 0.76 / Movie #1: 0.76
Minimum - Maximum-0.21194004 - 1.76278007
Average (Standard dev.)0.05694631 (±0.22409908)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 358.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.41.41.4
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z358.400358.400358.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-150-1500
NX/NY/NZ301301151
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.2121.7630.057

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Supplemental data

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Segmentation: A subunit 2

AnnotationA subunit 2
Fileemd_5476_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Segmentation: E subunit 3

AnnotationE subunit 3
Fileemd_5476_msk_10.map
Projections & Slices
AxesZYX

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Segmentation: G subunit 1

AnnotationG subunit 1
Fileemd_5476_msk_11.map
Projections & Slices
AxesZYX

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Segmentation: G subunit 2

AnnotationG subunit 2
Fileemd_5476_msk_12.map
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Segmentation: G subunit 3

AnnotationG subunit 3
Fileemd_5476_msk_13.map
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Segmentation: H subunit

AnnotationH subunit
Fileemd_5476_msk_14.map
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Segmentation: a subunit

Annotationa subunit
Fileemd_5476_msk_15.map
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Segmentation: c-ring

Annotationc-ring
Fileemd_5476_msk_16.map
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Segmentation: d subunit

Annotationd subunit
Fileemd_5476_msk_17.map
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Segmentation: detergent/lipid plug

Annotationdetergent/lipid plug
Fileemd_5476_msk_18.map
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Segmentation: A subunit 1

AnnotationA subunit 1
Fileemd_5476_msk_19.map
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Segmentation: A subunit 3

AnnotationA subunit 3
Fileemd_5476_msk_2.map
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Segmentation: B subunit 1

AnnotationB subunit 1
Fileemd_5476_msk_3.map
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Segmentation: B subunit 2

AnnotationB subunit 2
Fileemd_5476_msk_4.map
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Segmentation: B subunit 3

AnnotationB subunit 3
Fileemd_5476_msk_5.map
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Segmentation: C subunit

AnnotationC subunit
Fileemd_5476_msk_6.map
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Segmentation: DF subcomplex

AnnotationDF subcomplex
Fileemd_5476_msk_7.map
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Segmentation: E subunit 1

AnnotationE subunit 1
Fileemd_5476_msk_8.map
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Segmentation: E subunit 2

AnnotationE subunit 2
Fileemd_5476_msk_9.map
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Sample components

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Entire : vacuolar-type ATPases

EntireName: vacuolar-type ATPases
Components
  • Sample: vacuolar-type ATPases
  • Protein or peptide: vacuolar-type ATPases

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Supramolecule #1000: vacuolar-type ATPases

SupramoleculeName: vacuolar-type ATPases / type: sample / ID: 1000
Oligomeric state: A3B3CDE3FG3HadcXc'Yc''Z complex where X, Y, and Z indicate unknown stoichiometry
Number unique components: 1
Molecular weightExperimental: 900 KDa / Theoretical: 900 KDa / Method: Gel filtration

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Macromolecule #1: vacuolar-type ATPases

MacromoleculeName: vacuolar-type ATPases / type: protein_or_peptide / ID: 1 / Name.synonym: V-ATPase / Details: Detergent solubilized protein complex / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: SABY31 / synonym: Baker's yeast / Organelle: Intracellular membranes / Location in cell: Intracellular membranes
Molecular weightExperimental: 900 KDa / Theoretical: 900 KDa

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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

Concentration2.5 mg/mL
BufferpH: 7.4
Details: 50 mM Tris-HCl, 150 mM NaCl, 0.03% w/v dodecylmaltoside
GridDetails: Quantifoil R2/2 glow discharged in air
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III / Method: Blot for 20 seconds before freezing

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Alignment procedureLegacy - Astigmatism: Manually corrected by inspecting FFT
DateJan 1, 2011
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 1000 / Average electron dose: 12 e/Å2 / Bits/pixel: 8
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.0 Å / Resolution method: OTHER / Software - Name: Search_Fspace, Refine_Fspace, Build_Fspace / Number images used: 34448
Detailsparticles selected manually with Ximdisp

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