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- EMDB-1590: Structure of the Manduca sexta V-ATPase by cryo-electron microscopy -

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Basic information

Entry
Database: EMDB / ID: EMD-1590
TitleStructure of the Manduca sexta V-ATPase by cryo-electron microscopy
Map dataManduca sexta vacuolar ATPase
Sample
  • Sample: Manduca sexta vacuolar ATPase complex
  • Organelle or cellular component: membrane proton pump
KeywordsH-ATPase / V-ATPase / cryo-electron microscopy / vacuolar membrane
Biological speciesManduca sexta (tobacco hornworm)
Methodsingle particle reconstruction / cryo EM / Resolution: 17.0 Å
AuthorsMuench SP / Huss M / Phillips C / Song CF / Wieczorek H / Trinick J / Harrison MA
CitationJournal: J Mol Biol / Year: 2009
Title: Cryo-electron microscopy of the vacuolar ATPase motor reveals its mechanical and regulatory complexity.
Authors: Stephen P Muench / Markus Huss / Chun Feng Song / Clair Phillips / Helmut Wieczorek / John Trinick / Michael A Harrison /
Abstract: The vacuolar H+-ATPase (V-ATPase) is an ATP-driven rotary molecular motor that is a transmembrane proton pump in all eukaryotic cells. Although its activity is fundamental to many physiological ...The vacuolar H+-ATPase (V-ATPase) is an ATP-driven rotary molecular motor that is a transmembrane proton pump in all eukaryotic cells. Although its activity is fundamental to many physiological processes, our understanding of the structure and mechanism of the V-ATPase is poor. Using cryo-electron microscopy of the tobacco hornworm (Manduca sexta) enzyme, we have calculated the first 3D reconstruction of the intact pump in its native state. The resolution of 16.5 A is significantly higher than that of previous cryo-electron microscopy models of either V-ATPase or the related F1F0-ATPase. A network of four stalk structures connecting the V1 catalytic domain and the V0 membrane domain is now fully resolved, demonstrating substantially greater complexity than that found in the F-ATPase. Three peripheral stator stalks connect these domains to a horizontal collar that partly encircles the region between V1 and V0. The fourth stalk is a central axle that connects to V0 but makes minimal contact with V1. Several subunit crystal structures can be fit accurately into the reconstruction. The model thus provides new insights into the organisation of key components involved in mechanical coupling between the domains and regulation of activity.
History
DepositionJan 6, 2009-
Header (metadata) releaseJan 7, 2009-
Map releaseApr 1, 2009-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1590.gif

Downloads & links

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Map

FileDownload / File: emd_1590.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationManduca sexta vacuolar ATPase
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.36 Å/pix.
x 100 pix.
= 436. Å		4.36 Å/pix.
x 100 pix.
= 436. Å		4.36 Å/pix.
x 100 pix.
= 436. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.36 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 0.05 / Movie #1: 0.04
Minimum - Maximum-0.0450027 - 0.53537
Average (Standard dev.)0.00011227 (±0.0319842)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-50-49-50
Dimensions100100100
Spacing100100100
CellA=B=C: 436 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.364.364.36
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z436.000436.000436.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-165-165-40
NX/NY/NZ33033081
MAP C/R/S123
start NC/NR/NS-49-50-50
NC/NR/NS100100100
D min/max/mean-0.0450.5350.000

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Supplemental data

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Sample components

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Entire : Manduca sexta vacuolar ATPase complex

EntireName: Manduca sexta vacuolar ATPase complex
Components
  • Sample: Manduca sexta vacuolar ATPase complex
  • Organelle or cellular component: membrane proton pump

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Supramolecule #1000: Manduca sexta vacuolar ATPase complex

SupramoleculeName: Manduca sexta vacuolar ATPase complex / type: sample / ID: 1000 / Details: The sample was monodisperse / Oligomeric state: monomer / Number unique components: 1
Molecular weightExperimental: 900 KDa / Theoretical: 900 KDa

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Supramolecule #1: membrane proton pump

SupramoleculeName: membrane proton pump / type: organelle_or_cellular_component / ID: 1 / Name.synonym: V-ATPase / Oligomeric state: monomer / Recombinant expression: No
Source (natural)Organism: Manduca sexta (tobacco hornworm) / synonym: tobacco hornworm / Tissue: midgut

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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

Concentration2.0 mg/mL
BufferpH: 8.1
Details: 150 mM NaCl, 9.6 mM B mercaptoethanol, 20 mM TrisHCl. Solubilised in C12E10 detergent
GridDetails: 300 mesh Cu Lacey grid
VitrificationCryogen name: ETHANE / Chamber temperature: 22 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: Computer controlled blotting device
Method: A small vial of ethane was placed inside a larger liquid nitrogen reservoir. 3ul of protein sample was then applied to a lacey grid which had been glow discharged for 30 seconds prior to use. ...Method: A small vial of ethane was placed inside a larger liquid nitrogen reservoir. 3ul of protein sample was then applied to a lacey grid which had been glow discharged for 30 seconds prior to use. The grid was then blotted (1.6 seconds) and quickly frozen in liquid ethane using a computer operated device as described in White et al., 2003, J. Struct, Biol 144 246-252

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 69000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Gatan side entry cryo holder / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 22 K
Alignment procedureLegacy - Astigmatism: astigmatism corrected at 100,000 magnification
DateNov 10, 2007
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Digitization - Sampling interval: 15.0 µm / Number real images: 320 / Average electron dose: 15 e/Å2
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: phase flipping each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Imagic Eman / Number images used: 11742
DetailsParticles were picked using BOXER

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