Single-molecule FRET histograms were built based on the data arising from molecules containing both active donor and acceptor fluorophores (>100 molecules, except for the histograms shown in Figure 1F corresponding to the 15–60 min times, which are based on 50–60 molecules). All frames of each FRET trajectory (prior the first fluorophore bleaching event) were used to make single-molecule FRET histograms, where each frame yields a count in the histograms.