Fig. 5: Reconstruction and comparison of SNARE complexes and sub-complexes.

A Signal subtraction and local refinement in CryoSPARC⁸⁰ produced reconstructions of four different ɑ-SNAP—SNARE subcomplexes. In each case, the density of the SNARE proteins in the core of the subcomplex was sufficiently well-defined to assign the SNARE domain identity, directionality, and register. B Crystal structures of the neuronal ternary SNARE complex (PDB ID 1N7S⁵⁴) and the 2:2 syntaxin—SNAP-25 complex (PDB ID 1JTH³⁴) were superimposed using UCSF ChimeraX⁸⁴ with cryo-EM structures from this work of the tetrameric syntaxin H3 sub-complex from the sx20S, the 2:1 syntaxin—SNAP-25 sub-complex from the 21bin20S complex, and the 2:2 syntaxin—SNAP-25 sub-complex within the 22bin20S complex. These structures show similar quaternary structure, maintaining the same register and similar twist in the central SNARE layers. The SNARE complexes and sub-complexes within their respective 20S complexes are somewhat untwisted at the N- and C-terminal ends relative to the crystal structures of the SNARE complexes alone, likely due to a combination of the absence of crystal packing as well as interactions with ɑ-SNAPs and NSF. C Quantitative comparison of individual SNARE domain ternary structure. Each pixel in a pair of matrices represents the mean Cɑ RMSD (Å, left) and corresponding standard deviation (right) between two instances of a given SNARE domain in two different structures. Only Cɑ atoms shared by all structures were considered. The same color scale is preserved across all comparisons. The mean Cɑ RMSD generally varies between 1 and 2 Å depending on the specific pair. D Individual Cɑ RMSDs mapped to their corresponding SNARE domain structures, aligned. Core regions of the complexes are structurally similar, but unwinding near the SNARE domain termini leads to larger deviations, particularly near the N-termini where NSF engages one of the N-terminal segments leading into a SNARE domain (SNAP-25 or syntaxin).