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. 2025 Jan 2;16:250. doi: 10.1038/s41467-024-55531-0

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© The Author(s) 2025

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

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Fig. 2 — a Procedure of the smFRET assay of SNARE disassembly by NSF using TIRF microscope. b TIRF microscopy images FRET pair, Cy3 and Cy5 fluorescence channels representing SNARE-nanodisc after immobilisation along with a pictorial representation of nanodisc present on the glass slide before and after the disassembly by NSF. c–g Single-molecule trajectories of the NStx-Cy3 (green), NSN25-Cy5 (red) and FRET (dark grey) collected under the ALEX cycle between 532 nm and 640 nm lasers during the disassemble. Fl., fluorescence, Arb., arbitrary. c a representative trace showing both the NStx-Cy3 chain and NSN25-Cy5 chain disassembling at the same time. d a representative trace where the NStx-Cy3 chain disassembles first, followed by the NSN25-Cy5 chain. e a representative trace where the NSN25-Cy5 chain disassembles first, followed by the NStxCy3 chain. f a representative trace where a separation of the NStx-Cy3 chain is observed, followed by NStx-Cy3 disassemble first and then the NSN25-Cy5 chain. g a representative trace where a separation of the NSN25-Cy5 chain is observed, followed by NSN25-Cy5 disassemble first and then the NStx-Cy3 chain. h Classification of the single molecule traces based on the chain disassembly time (n = 11, each n represents an independent SNARE disassembly assay), data shown as mean ± SD. i, j Graphs showing the average lifetime of the chain separated state before the chain disassembly, 0.30 ± 0.03 seconds in case of traces (f) and 0.24 ± 0.01 s in case of traces (g). The dotted lines show the FRET separation and chain disassembly in traces (c–g).