Preparation of the nanodisc-20S particle was performed the same as previously described (30). The only difference was that we used more proteins to assemble the nanodisc-20S particle to achieve high concentration. The final sample volume was about 50 μl at a concentration of approximately 5 mg/ml. For the N-terminally truncated SNARE complex, the N-terminal sequence before the two amino acids leading up to layer −7 of each SNARE protein was deleted. The amino acid sequences of the NSF and SNARE proteins used in this study were listed in table S3.

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