|Entry||Database: EMDB / ID: 6204|
|Title||Structure of ATP-bound N-ethylmaleimide sensitive factor determined by single particle cryoelectron microscopy|
|Map data||Map of ATP-bound N-ethylmaleimide sensitive factor. This map is unsharpened and unfiltered. The map was normalized using the program MAPMAN.|
|Sample||ATP-bound N-ethylmaleimide sensitive factor:|
N-ethylmaleimide sensitive factor
|Keywords||ATPases associated with diverse cellular activities|
|Function / homology||Aspartate decarboxylase-like domain superfamily / CDC48, domain 2 / AAA-protein family signature. / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48), N-terminal domain / ATPase, AAA-type, conserved site / ATPase, AAA-type, core / AAA+ ATPase domain / CDC48, N-terminal subdomain / ATPase family associated with various cellular activities (AAA) ...Aspartate decarboxylase-like domain superfamily / CDC48, domain 2 / AAA-protein family signature. / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48), N-terminal domain / ATPase, AAA-type, conserved site / ATPase, AAA-type, core / AAA+ ATPase domain / CDC48, N-terminal subdomain / ATPase family associated with various cellular activities (AAA) / CDC48 domain 2-like superfamily / Vesicle-fusing ATPase / P-loop containing nucleoside triphosphate hydrolase / SNARE complex disassembly / vesicle-fusing ATPase / positive regulation of receptor recycling / syntaxin-1 binding / ionotropic glutamate receptor binding / SNARE binding / potassium ion transport / ATPase activity, coupled / PDZ domain binding / positive regulation of protein catabolic process / intracellular protein transport / midbody / ATPase activity / protein-containing complex binding / myelin sheath / protein kinase binding / ATP binding / identical protein binding / plasma membrane / metal ion binding / cytosol / Vesicle-fusing ATPase|
Function and homology information
|Source||Cricetulus griseus (Chinese hamster)|
|Method||single particle reconstruction / cryo EM / 4.2 Å resolution|
|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
|Validation Report||PDB-ID: 3j94|
SummaryFull reportAbout validation report
|Date||Deposition: Dec 4, 2014 / Header (metadata) release: Jan 28, 2015 / Map release: Jan 28, 2015 / Last update: Feb 11, 2015|
|Structure viewer||EM map: |
Downloads & links
|File||emd_6204.map.gz (map file in CCP4 format, 65537 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.2156 Å|
CCP4 map header:
-Entire ATP-bound N-ethylmaleimide sensitive factor
|Entire||Name: ATP-bound N-ethylmaleimide sensitive factor / Details: Wild type full-length construct. / 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: Feb 28, 2014|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 26.4 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: 50781|
Details: 3D classification, refinement, and reconstruction were performed using RELION.
|3D reconstruction||Software: RELION / CTF correction: Each particle|
Details: Every image is the sum of 30 frames recorded using the K2 Summit. The final reconstruction was calculated from images summed from frames #2-#18.
Resolution: 4.2 Å / Resolution method: FSC 0.143, gold-standard
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