|Entry||Database: PDB / ID: 3j94|
|Title||Structure of ATP-bound N-ethylmaleimide sensitive factor determined by single particle cryoelectron microscopy|
|Keywords||HYDROLASE / 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
AAA+ lid domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48), N-terminal domain / ATPase family associated with various cellular activities (AAA) / AAA ATPase, AAA+ lid domain / Vesicle-fusing ATPase / CDC48 domain 2-like superfamily / P-loop containing nucleoside triphosphate hydrolase / Aspartate decarboxylase-like domain superfamily ...AAA+ lid domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48), N-terminal domain / ATPase family associated with various cellular activities (AAA) / AAA ATPase, AAA+ lid domain / Vesicle-fusing ATPase / CDC48 domain 2-like superfamily / P-loop containing nucleoside triphosphate hydrolase / Aspartate decarboxylase-like domain superfamily / CDC48, domain 2 / ATPase, AAA-type, conserved site / ATPase, AAA-type, core / AAA+ ATPase domain
|Biological species||Cricetulus griseus (Chinese hamster)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å|
|Authors||Zhao, M. / Wu, S. / Cheng, Y. / Brunger, A.T.|
|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.
SummaryFull reportAbout validation report
|Structure viewer||Molecule: |
Downloads & links
A: Vesicle-fusing ATPase
B: Vesicle-fusing ATPase
C: Vesicle-fusing ATPase
D: Vesicle-fusing ATPase
E: Vesicle-fusing ATPase
F: Vesicle-fusing ATPase
Mass: 82907.430 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cricetulus griseus (Chinese hamster) / Gene: NSF / Production host: Escherichia coli (E. coli) / References: UniProt: P18708, vesicle-fusing ATPase
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Component||Name: ATP-bound N-ethylmaleimide sensitive factor / Type: COMPLEX / Details: hexamer|
|Molecular weight||Value: 0.5 MDa / Experimental value: NO|
|Buffer solution||Name: 50 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, 1 mM ATP, 1 mM DTT, 0.05% v/v Nonident P-40|
Details: 50 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, 1 mM ATP, 1 mM DTT, 0.05% v/v Nonident P-40
|Specimen||Conc.: 15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Details: Holey carbon on top of 400 mesh copper grid|
|Vitrification||Instrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Temp: 90 K / Humidity: 100 %|
Details: Blot for 3.5 seconds before plunging into liquid ethane (FEI VITROBOT MARK I).
Method: Blot for 3.5 seconds before plunging
-Electron microscopy imaging
Model: Tecnai Polara / Image courtesy: FEI Company
|Microscopy||Model: FEI POLARA 300 / Date: Feb 28, 2014|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 31000 X / Nominal defocus max: -2800 nm / Nominal defocus min: -1800 nm / Cs: 2.3 mm / Camera length: 0 mm|
|Specimen holder||Model: OTHER / Specimen holder type: unspecified|
|Image recording||Electron dose: 26.4 e/Å2 / Film or detector model: GATAN K2 (4k x 4k)|
|Radiation||Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray|
|Radiation wavelength||Relative weight: 1|
|CTF correction||Details: Each particle|
|Symmetry||Point symmetry: C1 (asymmetric)|
|3D reconstruction||Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50781 / Nominal pixel size: 1.2156 Å / Actual pixel size: 1.2156 Å|
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. (Single particle details: 3D classification, ...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. (Single particle details: 3D classification, refinement, and reconstruction were performed using RELION) (Single particle--Applied symmetry: C1)
Symmetry type: POINT
|Atomic model building||Protocol: FLEXIBLE FIT / Space: RECIPROCAL / Target criteria: R-factor|
Details: REFINEMENT PROTOCOL--flexible DETAILS--D2 domain was from crystal structure 1NSF. D1 domain was built de novo.
|Atomic model building||PDB-ID: 1NSF|
Pdb chain-ID: A
|Refinement||Resolution: 4.2→311.194 Å / SU ML: 0.78 / σ(F): 0.12 / Phase error: 37.83 / Stereochemistry target values: ML|
|Solvent computation||Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL|
|Displacement parameters||Biso max: 375.18 Å2 / Biso mean: 200.7475 Å2 / Biso min: 120.8 Å2|
|Refinement step||Cycle: LAST / Resolution: 4.2→311.194 Å|
|Refine LS restraints|
|LS refinement shell|
Refinement-ID: ELECTRON MICROSCOPY / Total num. of bins used: 30
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