Extended Data Fig. 8: Validating the behaviour of dyes used for smFRET.

a, The 50-ns molecular dynamics simulations of fluorophore tumbling on the BG505 sgp140 SOSIP.664 trimer (4ZMJ) shows that dyes in V1 and V4 are far from the viral membrane. Molecular dynamics simulation was performed to account for movements of loops, enzymatic labelling tags, linkers and dyes to describe the possible dye tumbling space within 50 ns. The sampled space was docked into the approximately 20 Å structure of the HIV-1 virus Env spike determined by cryo-electron tomography⁴. A 50-ns molecular dynamics simulation is not temporally comparable to the time resolution of single-molecule imaging at 40 ms, or the timescale of observed conformational changes of Env (milliseconds to seconds). b–d, Conformational properties of the HIV-1_BG505 Env remain highly similar when the dyes are flipped. b, Reference FRET histograms of HIV-1_BG505 that carries Cy3B in V1 and Cy5 in V4, in unliganded form (from Fig. 1e), in the presence of PGT151 (from Fig. 1h) or in the presence of sCD4_D1D2–Igαtp (from Fig. 2b). c, FRET histograms of HIV-1_BG505 Env that carries Cy5 in V1 and Cy3B in V4 (see Methods), in the absence and in the presence of 10 μg ml⁻¹ PGT151 or 10 μg ml⁻¹ sCD4_D1D2–Igαtp, respectively. d, Overlaid conformational landscapes of HIV-1_BG505 Env labelled as in c with flipped dyes (green), compared to HIV-1_BG505 Env labelled as in b (red). FRET population histograms represent mean ± s.e.m., determined from three independent populations of smFRET traces.