Fig. 4. Syntaxin anchored SNARE-nanodisc disassembly by NSF monitored using three-colour single-molecule FRET.

a Procedure of the smFRET assay of SNARE disassembly by NSF using TIRF microscope. b Pictorial representation of complexes on the glass slide before and after the disassembly by NSF. c Percentage of spots detected in the NVamp soluble-Cy3 channel before and after the disassembly. p = **< 0.01, two-sided paired t test, n = 5, individual experiments from multiple sample preparations. Values are mean ± SD. d Relative percentage of the classified of the traces based on the chain disassembly time and separation. n = 4, each n is data from an independent sample preparation with 2-3 experiments from each sample. Data is shown as mean ± SD. e–g Single-molecule trajectories of the NVamp-Cy3 (green), NSN25-Cy5 (red) and NStx-Cy7 (dark grey) during the disassembly. e a representative trace showing NVamp and NSN25 disassemble from NStx together at the same time. f a representative trace where Stx separation from NVamp-Cy3 and NSN25-Cy5 is observed before NVamp and NSN25 disassemble from NStx. g a representative trace where NVamp-Cy3 disassembles first, followed by NSN25-Cy5. h Cumulative distribution of the lifetime of the NVamp-Cy3 and NSN25-Cy5 separated state (n = 43 traces). i Pictorial representation of three major pathways of SNARE disassembly by NSF under our experimental design. I- Pathway showing NStx disassemble from NVamp and NSN25 first and vice versa (36%, n = 73 traces) and II-Pathway where a transient state with NStx separated from NVamp and NSN25 is observed before NStx disassembles (20.5%, n = 43 traces). III- Pathway showing NVamp disassembles first, followed by NSN25 (38.5%, n = 78 traces).