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- EMDB-2328: Structure of the bacterial V-ATPase from Thermus thermophilius -

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Basic information

Entry
Database: EMDB / ID: EMD-2328
TitleStructure of the bacterial V-ATPase from Thermus thermophilius
Map dataReconstruction of the bacterial V-ATPase
Sample
  • Sample: Bacterial V-type ATPase
  • Protein or peptide: V-ATPase
Keywordsbacterial V-ATPase / rotary ATPase
Biological speciesThermus thermophilus (bacteria)
Methodelectron crystallography / negative staining / Resolution: 18.0 Å
AuthorsTani K / Arthur CP / Tamakoshi M / Yokoyama K / Mitsuoka K / Fujiyoshi Y / Gerle C
CitationJournal: Microscopy (Oxf) / Year: 2013
Title: Visualization of two distinct states of disassembly in the bacterial V-ATPase from Thermus thermophilus.
Authors: Kazutoshi Tani / Christopher P Arthur / Masatada Tamakoshi / Ken Yokoyama / Kaoru Mitsuoka / Yoshinori Fujiyoshi / Christoph Gerle /
Abstract: V-ATPases are multisubunit, membrane-bound, energy-converting, cellular machines whose assembly and disassembly is innately connected to their activity in vivo. In vitro V-ATPases show a propensity ...V-ATPases are multisubunit, membrane-bound, energy-converting, cellular machines whose assembly and disassembly is innately connected to their activity in vivo. In vitro V-ATPases show a propensity for disassembly that greatly complicates their functional, and, in particular, structural characterization. Direct structural evidence for early stages of their disassembly has not been reported yet. We analyzed the structure of the V-ATPase from Thermus thermophilus in a single negatively stained two-dimensional (2-D) crystal both by electron tomography and by electron crystallography. Our analysis demonstrated that for 2-D crystals of fragile macromolecular complexes, which are too heterogenous or too few for the merging of image data from many crystals, single-crystal 3-D reconstructions by electron tomography and electron crystallography are expedient tools of analysis. The asymmetric unit in the 2-D crystal lattice contains two different V-ATPase complexes that appear to be in an early stage of disassembly and with either one or both peripheral stalks not being visualized, suggesting the involvement of the peripheral stalks in early stages of disassembly.
History
DepositionMar 10, 2013-
Header (metadata) releaseMar 20, 2013-
Map releaseApr 17, 2013-
UpdateAug 28, 2013-
Current statusAug 28, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

2328_emd-232...

emd-2328-VA_...

Downloads & links

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Map

FileDownload / File: emd_2328.map.gz / Format: CCP4 / Size: 476.6 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the bacterial V-ATPase
Voxel sizeX: 4.296 Å / Y: 4.4 Å / Z: 4.166 Å
Density
Contour LevelBy AUTHOR: 0.4 / Movie #1: 0.4
Minimum - Maximum-3.48900008 - 6.74800014
Average (Standard dev.)-0.00587434 (±0.98739201)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-27-15-36
Dimensions553173
Spacing305472
CellA: 231.984 Å / B: 132.0 Å / C: 299.952 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.2964.44.166
M x/y/z543072
origin x/y/z0.0000.0000.000
length x/y/z231.984132.000299.952
α/β/γ90.00090.00090.000
start NX/NY/NZ-27-15-36
NX/NY/NZ553173
MAP C/R/S213
start NC/NR/NS-15-27-36
NC/NR/NS315573
D min/max/mean-3.4896.748-0.006

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Supplemental data

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Sample components

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Entire : Bacterial V-type ATPase

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

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Supramolecule #1000: Bacterial V-type ATPase

SupramoleculeName: Bacterial V-type ATPase / type: sample / ID: 1000 / Number unique components: 1

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Macromolecule #1: V-ATPase

MacromoleculeName: V-ATPase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Thermus thermophilus (bacteria) / Location in cell: Plasma membrane

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Experimental details

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Structure determination

Methodnegative staining
Processingelectron crystallography
Aggregation state2D array

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Sample preparation

Concentration1 mg/mL
StainingType: NEGATIVE
Details: A 2.5 ul sample was transferred to a glow-discharged carbon coated copper grid. After 1 min, the drop was blotted with a slice of filter paper, and then the grid was washed with 2.5 ul water ...Details: A 2.5 ul sample was transferred to a glow-discharged carbon coated copper grid. After 1 min, the drop was blotted with a slice of filter paper, and then the grid was washed with 2.5 ul water to avoid the precipitation caused by mixing phosphate buffer with uranyl acetate. Subsequently, the sample was stained with 2.5 ul of 2% uranyl acetate and air-dried.
GridDetails: a glow-discharged carbon coated copper grid
VitrificationCryogen name: NONE / Instrument: OTHER
DetailsCrystals grown by dialysis
Crystal formationDetails: Crystals grown by dialysis

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.27 µm / Nominal defocus min: 1.4 µm / Nominal magnification: 40000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle min: -55 / Tilt angle max: 55 / Tilt series - Axis1 - Min angle: -55 ° / Tilt series - Axis1 - Max angle: 55 °
DateNov 28, 2007
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN / Digitization - Sampling interval: 15 µm / Number real images: 212
Details: Every image was recorded by 2Kx2K CCD camera (Gatan)
Bits/pixel: 16
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

Crystal parametersUnit cell - A: 232 Å / Unit cell - B: 132 Å / Unit cell - C: 300 Å / Unit cell - γ: 90.0 ° / Unit cell - α: 90.0 ° / Unit cell - β: 90.0 ° / Plane group: P 1
Final reconstructionResolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: OTHER / Software - Name: MRC
DetailsImages were processed using MRC package

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