|Entry||Database: EMDB / ID: 2781|
|Title||Structure of the vacuolar H+-ATPase rotary motor at subnanometer resolution|
|Map data||V-ATPase reconstruction|
|Sample||Manduca sexta vacuolar ATPase complex:|
|Keywords||Rotary ATPase / vacuolar ATPase|
|Source||Manduca sexta (tobacco hornworm)|
|Method||single particle reconstruction / cryo EM / 9.4 Å resolution|
|Authors||Rawson S / Phillips C / Huss M / Tiburcy F / Wieczorek H / Trinick J / Harrison MA / Muench SP|
|Citation||Journal: Structure / Year: 2015|
Title: Structure of the vacuolar H+-ATPase rotary motor reveals new mechanistic insights.
Authors: Shaun Rawson / Clair Phillips / Markus Huss / Felix Tiburcy / Helmut Wieczorek / John Trinick / Michael A Harrison / Stephen P Muench
Abstract: Vacuolar H(+)-ATPases are multisubunit complexes that operate with rotary mechanics and are essential for membrane proton transport throughout eukaryotes. Here we report a ∼ 1 nm resolution ...Vacuolar H(+)-ATPases are multisubunit complexes that operate with rotary mechanics and are essential for membrane proton transport throughout eukaryotes. Here we report a ∼ 1 nm resolution reconstruction of a V-ATPase in a different conformational state from that previously reported for a lower-resolution yeast model. The stator network of the V-ATPase (and by implication that of other rotary ATPases) does not change conformation in different catalytic states, and hence must be relatively rigid. We also demonstrate that a conserved bearing in the catalytic domain is electrostatic, contributing to the extraordinarily high efficiency of rotary ATPases. Analysis of the rotor axle/membrane pump interface suggests how rotary ATPases accommodate different c ring stoichiometries while maintaining high efficiency. The model provides evidence for a half channel in the proton pump, supporting theoretical models of ion translocation. Our refined model therefore provides new insights into the structure and mechanics of the V-ATPases.
|Date||Deposition: Sep 5, 2014 / Header (metadata) release: Sep 24, 2014 / Map release: Feb 18, 2015 / Last update: Feb 17, 2016|
|Structure viewer||EM map: |
Downloads & links
|File||emd_2781.map.gz (map file in CCP4 format, 128001 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.35 Å|
CCP4 map header:
-Entire Manduca sexta vacuolar ATPase complex
|Entire||Name: Manduca sexta vacuolar ATPase complex / Details: monodisperse / Number of components: 1 / Oligomeric State: monomeric|
|Mass||Experimental: 900 kDa / Measured by: mass spec|
-Component #1: cellular-component, Vacuolar ATPase
|Cellular-component||Name: Vacuolar ATPaseV-ATPase / a.k.a: V-ATPase / Oligomeric Details: monomer / Recombinant expression: No / Number of Copies: 1|
|Mass||Experimental: 900 kDa|
|Source||Species: Manduca sexta (tobacco hornworm)|
|Specimen||Specimen state: particle / Method: cryo EM|
|Sample solution||Specimen conc.: 0.025 mg/ml|
Buffer solution: 150 mM NaCl, 20 mM Tris-HCl, 9.6 mM 2-mercaptoethanol, 0.01% C12E10
|Support film||400 mesh Quantifoil R2.0/2.0 grids with thin carbon (10 nm) coating, glow discharged in air.|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 120 K / Humidity: 100 % / Method: Grids were blotted for 7.5 seconds|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS / Date: Feb 27, 2014 / Details: Collected with FEI EPU software|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 60 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 103704 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 5500 nm|
|Specimen Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Camera||Detector: FEI FALCON II (4k x 4k)|
|Image acquisition||Number of digital images: 1366|
Details: Each micrograph is sum of 34 frames recorded by direct detector.
|Processing||Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 6714 / Details: Standard procedures in RELION1.3|
|3D reconstruction||Algorithm: Maximum Likelihood / Software: Relion / CTF correction: Relion|
Details: Maximum likelihood in Relion using 3D auto-refine. The particles were handpicked in BOXER
Resolution: 9.4 Å / Resolution method: FSC 0.143, gold-standard
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