Fig. 3. Comparison of smFRET data to values predicted from crystal structure of SurA.

The crystal structure of SurA indicating the available volumes calculated for each dye label at residues a 85 and 193 (core-P1 distance probes); b 85 and 301 (core-P2 distance probes) and c 193 and 301 (P1-P2 distance probes). d–f Predicted E_FRET between each pair of dyes for the crystal structure of SurA (black line) and the observed E_FRET distributions (green, red or blue lines) independently fitted to two Gaussians (pink/yellow). g–i BVA reveals high variance for all three dye pairs. The average values of the measured variance for the E_FRET values (white filled circles) lie above the expected shot-noise limited standard deviation (black arc) indicating dynamics on a timescale faster than the duration of the bursts (here sub-ms). Predicted E_FRET distributions d–f were calculated from the SurA crystal structure (PDB 1M5Y²⁷) using distance distributions generated by the MtsslWizard plugin for PyMOL¹⁰³, which takes into account both the location of the dyes and the flexibility of the dye linkers³⁹. Note that all dyes can be considered as freely rotating, as manifested by their low anisotropy (Supplementary Table 2), hence changes in E_FRET can be translated to distance variations. Samples contained ~50 pM labelled SurA variant in 50 mM Tris-HCl, pH 8.0, 25 °C.