|Entry||Database: EMDB / ID: 8944|
|Title||20S supercomplex consisting of linked neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)|
|Map data||20S supercomplex with linked SNARE|
|Sample||20S Supercomplex with linked SNARE complex:|
|Method||single particle reconstruction / cryo EM / 7 Å resolution|
|Authors||Zhao M / Choi UB / Brunger AT|
|Citation||Journal: Elife / Year: 2018|
Title: NSF-mediated disassembly of on- and off-pathway SNARE complexes and inhibition by complexin.
Authors: Ucheor B Choi / Minglei Zhao / K Ian White / Richard A Pfuetzner / Luis Esquivies / Qiangjun Zhou / Axel T Brunger
Abstract: SNARE complex disassembly by the ATPase NSF is essential for neurotransmitter release and other membrane trafficking processes. We developed a single-molecule FRET assay to monitor repeated rounds of ...SNARE complex disassembly by the ATPase NSF is essential for neurotransmitter release and other membrane trafficking processes. We developed a single-molecule FRET assay to monitor repeated rounds of NSF-mediated disassembly and reassembly of individual SNARE complexes. For ternary neuronal SNARE complexes, disassembly proceeds in a single step within 100 msec. We observed short- (<0.32 s) and long-lived (≥0.32 s) disassembled states. The long-lived states represent fully disassembled SNARE complex, while the short-lived states correspond to failed disassembly or immediate reassembly. Either high ionic strength or decreased αSNAP concentration reduces the disassembly rate while increasing the frequency of short-lived states. NSF is also capable of disassembling anti-parallel ternary SNARE complexes, implicating it in quality control. Finally, complexin-1 competes with αSNAP binding to the SNARE complex; addition of complexin-1 has an effect similar to that of decreasing the αSNAP concentration, possibly differentially regulating cis and trans SNARE complexes disassembly.
|Date||Deposition: Jul 6, 2018 / Header (metadata) release: Jul 25, 2018 / Map release: Jul 25, 2018 / Last update: Jul 25, 2018|
|Structure viewer||EM map: |
Downloads & links
|File||emd_8944.map.gz (map file in CCP4 format, 13501 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 2.02 Å|
CCP4 map header:
-Entire 20S Supercomplex with linked SNARE complex
|Entire||Name: 20S Supercomplex with linked SNARE complex / Number of components: 1|
-Component #1: protein, 20S Supercomplex with linked SNARE complex
|Protein||Name: 20S Supercomplex with linked SNARE complex / Recombinant expression: No|
|Source (engineered)||Expression System: Escherichia coli (E. coli)|
|Specimen||Specimen state: particle / Method: cryo EM|
|Sample solution||Specimen conc.: 15 mg/ml / pH: 8|
|Vitrification||Instrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 293 K / Humidity: 100 %|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 78 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Cs: 2.7 mm / Imaging mode: BRIGHT FIELD|
|Specimen Holder||Model: OTHER|
|Camera||Detector: GATAN K2 SUMMIT (4k x 4k)|
|Processing||Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 45194|
|3D reconstruction||Resolution: 7 Å / Resolution method: FSC 0.143 CUT-OFF|
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