Entire 20S supercomplex consisting of VAMP-7 SNARE complex, alpha-SNAP, ...
Entire
Name: 20S supercomplex consisting of VAMP-7 SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF) Number of components: 5 Oligomeric State: One hexamer of NSF + four alpha-SNAP molecules + one SNARE complex
Specimen conc.: 15 mg/ml Buffer solution: 50 mM Tris-Cl, 150 mM NaCl, 1 mM ATP, 1 mM EDTA, 1 mM DTT, 0.05% v/v Nonident P-40 pH: 8
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.
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Electron microscopy imaging
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
Imaging
Microscope: FEI POLARA 300 / Date: May 12, 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)
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Image acquisition
Image acquisition
Details: Gatan K2 Summit in super-resolution counting mode. Motion correction as described in Li et al. (2013) Nature Methods.
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Image processing
Processing
Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 32100 Details: 3D classification, refinement, and reconstruction were performed using RELION.
3D reconstruction
Software: RELION / CTF correction: Each particle / Resolution: 8 Å / Resolution method: FSC 0.143, gold-standard
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