RELRCSBRCSBRCSB2014-12-052015-01-282015-01-282015-02-11Structure of 20S supercomplex determined by single particle cryoelectron microscopy, state IIIbZhao MWu SZhou QVivona SCipriano DJCheng YBrunger ATvesicle traffickingZhao MWu SZhou QVivona SCipriano DJCheng YBrunger ATMechanistic insights into the recycling machine of the SNARE complexNATURE5186167201525581794doi:10.1038/nature141483j99FULLOVERLAP20S supercomplex consisting of truncated neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)20S supercomplex consisting of truncated neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)One hexamer of NSF + four alpha-SNAP molecules + one SNARE complex50.66N-ethylmaleimide sensitive factorCricetulus griseusChinese hamster0.0836hexamertrueEscherichia coliBL21-DE3-RILpROEX-1P18708alpha Soluble NSF Attachment ProteinRattus norvegicusRat0.0334trueEscherichia coliBL21(DE3)pJ414P54921Syntaxin-1ARattus norvegicusRat0.0081trueEscherichia coliBL21(DE3)pACYC-DUET-1P32851Synaptobrevin-2Rattus norvegicusRat0.0081trueEscherichia coliBL21(DE3)pACYC-DUET-1P63045Synaptosomal-associated protein 25Rattus norvegicusRat0.0161trueEscherichia coliBL21(DE3)pET-DUET-1P60881singleParticleparticle158.050 mM Tris-Cl, 150 mM NaCl, 1 mM AMPPNP, 1 mM EDTA, 1 mM DTT, 0.05% v/v Nonident P-40Holey carbon on top of 400 mesh copper gridETHANE10090FEI VITROBOT MARK IBlot for 3.5 seconds before plunging.FEI POLARA 300FLOOD BEAMBRIGHT FIELDFIELD EMISSION GUN3002.3-1.8-2.831000.0OTHER2014-01-28GATAN K2 (4k x 4k)44Gatan K2 Summit in super-resolution counting mode. Motion correction as described in Li et al. (2013) Nature Methods.3D classification, refinement, and reconstruction were performed using RELION.Each particle8.2OTHERRELION149911NSFAFLEXIBLE FITChimera, PHENIXD2 domain of NSF was from crystal structure 1NSF. D1 domain of NSF was from related entry EMD-6204. N domain of NSF was from crystal structure 1QCS. aSNAP was a homology model. SNARE complex was from crystal structure 1N7S.R-factorRECIPROCAL1QCSAFLEXIBLE FITChimera, PHENIXD2 domain of NSF was from crystal structure 1NSF. D1 domain of NSF was from related entry EMD-6204. N domain of NSF was from crystal structure 1QCS. aSNAP was a homology model. SNARE complex was from crystal structure 1N7S.R-factorRECIPROCAL1N7SABCDFLEXIBLE FITChimera, PHENIXD2 domain of NSF was from crystal structure 1NSF. D1 domain of NSF was from related entry EMD-6204. N domain of NSF was from crystal structure 1QCS. aSNAP was a homology model. SNARE complex was from crystal structure 1N7S.R-factorRECIPROCAL