Fig. 5.

Single-molecule FRET. Comparison of experimental (light brown wide bars) and calculated (black narrow bars) FRET efficiency histograms, using the interdye distances for the model structures, for a threshold number of photons (n_T) of (A) 50 and (B) 110 with a bin time (T_bin) of 2 ms. In the experimental histogram, the FRET efficiency of each bin was γ-corrected after subtraction of the average background photons (donor: 0.6 ms^-1; acceptor: 1.1 ms^-1) and the correction for leak of donor fluorescence into the acceptor channel (6%). The fluorescence bursts from the donor-only-labeled molecules (E < 0.18) were excluded and the data were prefiltered to remove bursts of photons in which either blinking or bleaching occurred. (C) Inferred model for an open state of ESCRT-I (Top) consisting of a continuum of conformations, in equilibrium with a closed state (Bottom) consisting of one or a few related conformations.