The available volume (AV) approach (39,40) was used to quantify the transfer efficiencies for each construct. For the Cy3/Cy5 FRET pair used here the parameters were assessed by measuring the dye and linker dimensions in VMD and these are shown in Supplementary Table S6. The distances between AV determined fluorophore mean positions were found for all frames. Transfer efficiencies were calculated based on the fluorophore distances using four approaches with different frames of the trajectories as input. Supplementary Figure S5 shows the average distance and average efficiency for all four approaches.