To build the model for the α-SNAP–SNARE subcomplex, on the basis of the visibility of the extended density corresponding to the longer C terminus of VAMP and the density map of several layers in the central part of the SNARE complex, the crystal structure of the neuronal synaptic fusion complex [PDB accession code: 1SFC; (2)] was docked into the density map by Chimera (45). The different residues between SNAP25B (the protein we used in this study) and SNAP25A (the protein in 1SFC) were manually mutated using Coot (46). The homology model of the Bos taurus α-SNAP was created using Modeller (47) with the crystal structure of the vesicular transport protein Sec17 [PDB accession code: 1QQE; (27)] as template and was docked into the map. Adjustments of backbones and side chains of the SNARE proteins and α-SNAP were performed manually on the basis of the density map in Coot. Then, real-space model refinement was performed in Phenix (48).

To build the model of the NSF-D1D2 part, the homology model of the D1 domain was created using Modeller with the crystal structure of the D1 domain of p97 [PDB accession code: 1E32; (49)] as template. The homology model was docked into the best copy of the six copies of D1 densities in the map by Chimera and manually adjusted in Coot. Then, the D1 model was docked into the other five copies of the D1 densities and further manually adjusted in Coot. The crystal structure of the D2 domain [PDB accession code: 1D2N; (26)] was docked into each of the six copies of the D2 densities. The linker between the D1 domain and the D2 domain was manually built in Coot. Then, the whole model was refined as the α-SNAP–SNARE subcomplex described above.

The atomic models were cross-validated according to previously described procedures (50). Briefly, atoms in the final model were randomly shifted by up to 0.2 Å, and the new model was then refined against one of two half-maps generated during the final 3D reconstruction. FSC values were calculated between the map generated from the resulting model and the two half-maps, as well as the averaged map of two half-maps. We did not observe remarkable separation between FSCwork and FSCfree, indicating that our model was not over-refined. The statistics of the geometries of the models were generated using MolProbity (51) and listed in table S1. All the figures were prepared in PyMOL (52) or Chimera (45).

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