|Entry||Database: EMDB / ID: EMD-6205|
|Title||Structure of ADP-bound N-ethylmaleimide sensitive factor determined by single particle cryoelectron microscopy|
|Sample||ADP-bound N-ethylmaleimide sensitive factor:|
N-ethylmaleimide sensitive factor
|Keywords||ATPases associated with diverse cellular activities|
|Function / homology|
Function and homology information
SNARE complex disassembly / vesicle-fusing ATPase / positive regulation of receptor recycling / syntaxin-1 binding / ionotropic glutamate receptor binding / SNARE binding / potassium ion transport / PDZ domain binding / positive regulation of protein catabolic process / intracellular protein transport ...SNARE complex disassembly / vesicle-fusing ATPase / positive regulation of receptor recycling / syntaxin-1 binding / ionotropic glutamate receptor binding / SNARE binding / potassium ion transport / PDZ domain binding / positive regulation of protein catabolic process / intracellular protein transport / midbody / ATPase activity / protein-containing complex binding / protein kinase binding / ATP binding / identical protein binding / plasma membrane / metal ion binding / cytosol
Aspartate decarboxylase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase / CDC48, domain 2 / ATPase, AAA-type, conserved site / AAA ATPase, AAA+ lid domain / Vesicle-fusing ATPase / ATPase, AAA-type, core / AAA+ ATPase domain / CDC48, N-terminal subdomain / CDC48 domain 2-like superfamily
|Biological species||Cricetulus griseus (Chinese hamster)|
|Method||single particle reconstruction / cryo EM / Resolution: 7.6 Å|
|Authors||Zhao M / Wu S / Zhou Q / Vivona S / Cipriano DJ / Cheng Y / Brunger AT|
|Citation||Journal: Nature / Year: 2015|
Title: Mechanistic insights into the recycling machine of the SNARE complex.
Authors: Minglei Zhao / Shenping Wu / Qiangjun Zhou / Sandro Vivona / Daniel J Cipriano / Yifan Cheng / Axel T Brunger /
Abstract: Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N- ...Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N-ethylmaleimide sensitive factor), together with SNAPs (soluble NSF attachment protein), disassembles the SNARE complex into its protein components, making individual SNAREs available for subsequent rounds of fusion. Here we report structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE (20S) supercomplex determined by single-particle electron cryomicroscopy at near-atomic to sub-nanometre resolution without imposing symmetry. Large, potentially force-generating, conformational differences exist between ATP- and ADP-bound NSF. The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains to pseudo four-fold symmetry of the SNARE complex. SNAPs interact with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism. The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes.
|Validation Report||PDB-ID: 3j95|
SummaryFull reportAbout validation report
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_6205.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 2.4312 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Supplemental map: EMD-6205 ADP NSF sharpened -479.map
-Entire ADP-bound N-ethylmaleimide sensitive factor
|Entire||Name: ADP-bound N-ethylmaleimide sensitive factor / Number of components: 1 / Oligomeric State: hexamer|
|Mass||Theoretical: 500 kDa|
-Component #1: protein, N-ethylmaleimide sensitive factor
|Protein||Name: N-ethylmaleimide sensitive factor / a.k.a: NSF / Oligomeric Details: hexamer / Recombinant expression: Yes / Number of Copies: 6|
|Mass||Theoretical: 83 kDa|
|Source||Species: Cricetulus griseus (Chinese hamster)|
|Source (engineered)||Expression System: Escherichia coli (E. coli) / Vector: pPROEX-1 / Strain: BL21(DE3)-RIL|
|External references||UniProt: Vesicle-fusing ATPase|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||Specimen conc.: 15 mg/mL|
Buffer solution: 50 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, 1 mM ATP, 1 mM DTT, 0.05% v/v Nonident P-40
|Support film||Holey carbon on top of 400 mesh copper grid|
|Vitrification||Instrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Temperature: 90 K / Humidity: 100 % / Method: Blot for 3.5 seconds before plunging.|
-Electron microscopy imaging
Model: Tecnai Polara / Image courtesy: FEI Company
|Imaging||Microscope: FEI POLARA 300 / Date: Jan 14, 2014|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 44 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 31000 X (nominal) / Cs: 2.3 mm / Imaging mode: BRIGHT FIELD / Defocus: -1800 - -2800 nm|
|Specimen Holder||Model: OTHER|
|Camera||Detector: GATAN K2 (4k x 4k)|
|Image acquisition||Details: Gatan K2 Summit in super-resolution counting mode. Motion correction as described in Li et al. (2013) Nature Methods.|
|Processing||Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 12830 |
Details: 3D classification, refinement, and reconstruction were performed using RELION.
|3D reconstruction||Software: RELION / CTF correction: Each particle / Resolution: 7.6 Å / Resolution method: FSC 0.143, gold-standard|
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