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- EMDB-1640: Structure of the V-ATPase of Saccharomyces cerevisiae at 2.5 nm r... -

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Basic information

Entry
Database: EMDB / ID: EMD-1640
TitleStructure of the V-ATPase of Saccharomyces cerevisiae at 2.5 nm resolution
Map dataV-ATPase of Sacharomyces cerevisiae
Sample
  • Sample: V-ATPase
  • Protein or peptide: V-ATPase
KeywordsV-ATPase / single particle / Stator
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsDiepholz M / Venzke D / Prinz S / Batisse C / Florchinger B / Rossle M / Svergun D / Bottcher B / Fethiere J
CitationJournal: Structure / Year: 2008
Title: A different conformation for EGC stator subcomplex in solution and in the assembled yeast V-ATPase: possible implications for regulatory disassembly.
Authors: Meikel Diepholz / David Venzke / Simone Prinz / Claire Batisse / Beate Flörchinger / Manfred Rössle / Dmitri I Svergun / Bettina Böttcher / James Féthière /
Abstract: Vacuolar ATPases (V-ATPases) are ATP-dependent proton pumps that maintain the acidity of cellular compartments. They are composed of a membrane-integrated proton-translocating V(0) and an extrinsic ...Vacuolar ATPases (V-ATPases) are ATP-dependent proton pumps that maintain the acidity of cellular compartments. They are composed of a membrane-integrated proton-translocating V(0) and an extrinsic cytoplasmic catalytic domain V(1), joined by several connecting subunits. To clarify the arrangement of these peripheral connections and their interrelation with other subunits of the holocomplex, we have determined the solution structures of isolated EG and EGC connecting subcomplexes by small angle X-ray scattering and the 3D map of the yeast V-ATPase by electron microscopy. In solution, EG forms a slightly kinked rod, which assembles with subunit C into an L-shaped structure. This model is supported by the microscopy data, which show three copies of EG with two of these linked by subunit C. However, the relative arrangement of the EG and C subunits in solution is more open than that in the holoenzyme, suggesting a conformational change of EGC during regulatory assembly and disassembly.
History
DepositionAug 3, 2009-
Header (metadata) releaseJun 11, 2010-
Map releaseJun 11, 2010-
UpdateJun 11, 2010-
Current statusJun 11, 2010Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

vatpase_emdb...

Downloads & links

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Map

FileDownload / File: emd_1640.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationV-ATPase of Sacharomyces cerevisiae
Voxel sizeX=Y=Z: 5.17 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.493566 - 0.977651
Average (Standard dev.)0.00408422 (±0.0519208)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 517 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.175.175.17
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z517.000517.000517.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean-0.4940.9780.004

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Supplemental data

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Sample components

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Entire : V-ATPase

EntireName: V-ATPase
Components
  • Sample: V-ATPase
  • Protein or peptide: V-ATPase

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Supramolecule #1000: V-ATPase

SupramoleculeName: V-ATPase / type: sample / ID: 1000 / Oligomeric state: A3B3CDE3FG3Hac'c''c(5-8)d / Number unique components: 1
Molecular weightTheoretical: 980 KDa

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Macromolecule #1: V-ATPase

MacromoleculeName: V-ATPase / type: protein_or_peptide / ID: 1 / Name.synonym: V-ATPase / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast / Tissue: Inner membranes / Cell: SBY119 / Organelle: Vacuoles / Location in cell: Vacuolar membranes
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.1
Details: Phosphate buffered Saline containing 0.1% Digitonin, 8% sucrose, 2% sorbitol, 2% glucose, Pefabloc SC (Roche), 13 tablets/l of complete EDTA-free Protease inhibitors
StainingType: NEGATIVE
Details: The sandwich technique (Golas et al., 2003) was used to prepare negatively stained V-ATPase samples. Briefly, purified V-ATPase at a concentration of 0.03 mg/ml was adsorbed on a carbon film ...Details: The sandwich technique (Golas et al., 2003) was used to prepare negatively stained V-ATPase samples. Briefly, purified V-ATPase at a concentration of 0.03 mg/ml was adsorbed on a carbon film layered on a mica support at the carbon/mica interface. Subsequently, the carbon film with adsorbed particles was floated over the staining solution (2% uranyl acetate). After attachment of a copper grid to the dry surface of the carbon, a second carbon film was floated over the same staining solution. The protein/carbon/grid assembly was picked up with a piece of newspaper, turned upside down, and immersed in the solution. A sandwich of two carbon layers with the protein particles trapped in between is created by picking up the second carbon layer with the grid.
GridDetails: 400 mesh copper grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal magnification: 27500
Sample stageSpecimen holder: Eucentric / Specimen holder model: SIDE ENTRY, EUCENTRIC
Alignment procedureLegacy - Astigmatism: Corrected at 200000 times magnification on graininess of carbon
Image recordingCategory: CCD / Film or detector model: GENERIC CCD / Digitization - Sampling interval: 14.22 µm / Number real images: 400
Details: Images were recorded on CCD, no scanning, sampling step size was adjusted to calibrated image size
Bits/pixel: 12
Tilt angle min0
Tilt angle max0

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC, SPIDER, EMAN
Details: Spider option BP 32F Back Projection - 3D, Sampled, Interpolated in Fourier space
Number images used: 16300

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