Extended Data Fig. 4: Quantifying the timing of transport initiation.

a, The precise timing of ligand injection was estimated by co-injecting a low concentration (0.5 nM) of Cy5-labelled 21-mer DNA duplex (Supplementary Methods) to the injected solution of interest, and measuring the increase in background fluorescence on the Cy5 channel in regions far from immobilized particles. The mean background fluorescence is shown, from a representative experiment in which LIV-BP^WT-containing liposomes that lack MhsT were imaged before and after (vertical dashed line) the injection of Cy5-labelled DNA tracer, with the exact time of injection determined by the midpoint of the step-like increase in fluorescence. Inset, zoomed-in view of the period immediately before and after injection. b, c, Representative single-molecule fluorescence (donor in green and acceptor in red) and FRET (blue) traces (b) and FRET contour plot (c) of encapsulated LIV-BP^WT from these experiments, demonstrating minimal changes in apparent FRET efficiency with co-injection of low concentrations of the Cy5 fluorophore. Experiments were performed at least three times with similar results.