(A) Experimental setup and structural model of the template complex. Some key mutations tested in this study are indicated by dots: red (phosphomimetic mutations) or gray (others) for Munc18-1, green for syntaxin, and orange for VAMP2. The helical hairpin of Munc18-1 domain 3a is highlighted in yellow. The NRD of syntaxin comprises an N-peptide (a.a. 1–26, see B), a three-helical Habc domain (27–146, deep salmon), and a linker region (147–199, brown). The structural model of the template complex is derived from a similar model of Vps33:Vam3:Nyv1 (Baker et al., 2015) by extending the N-terminal helix of the R-SNARE to the −7 layer, as justified herein. The NRD stabilizes the template complex, but its positioning in this model is arbitrary. See also Figure 2—figure supplement 1. (B) Crystal structure of closed syntaxin bound to Munc18-1 (PDB ID 3C98) (Misura et al., 2000). Highlighted are crosslinking sites (I187, R198, and L205), sites of mutations used to destabilize closed syntaxin (E76K, L165A, and E166A, green dots), and sites of phosphomimetic mutations (red dots and Y473). (C) Force-extension curves (FECs) obtained in the absence (#1) or presence (other FECs) of Munc18-1 in solution. Throughout the figures, all FECs are color coded in the same fashion: gray for pulling the initial purified SNARE complex, blue for subsequent pulls, black for relaxation, and red for holding the system at constant force. The states associated with different extensions (marked by green dashed lines as needed) are numbered as in Figure 2D. CTD transitions are indicated by gray ovals, NTD unfolding by gray arrows, t-SNARE unfolding by black arrows, syntaxin transitions by blue rectangles, and template complex transitions by blue ovals. See also Figure 2—figure supplement 2 and Figure 2—source datas 1 and 2 in Dataset 1. (D) Schematic diagrams of different states: 1, fully assembled SNARE complex; 2, half-zippered SNARE bundle; 3, t-SNARE complex; 4, fully unfolded SNARE motifs; 5, unfolded SNARE motifs with Munc18-1 bound; 6, partially closed syntaxin; 7, template complex; and 8, Munc18-1-bound assembled SNARE complex. In states 2 and 3, the t-SNARE complex is ordered up to the +4 layer (Gao et al., 2012; Ma et al., 2015; Zhang et al., 2016b). The states are numbered according to the same convention throughout the text and figures.