Extended Data Fig. 5: Anisotropic breathing motion of rhodopsin.

Comparison of the overall conformational changes in rhodopsin photoactivated for 1, 10 and 100 picoseconds. The difference electron density map (Fobs(1ps-light)-Fobs(dark) contoured at 4.2 rmsd) from the dataset of 1ps-illuminated rhodopsin superimposed on the rhodopsin dark state structure model (grey) (a-b) shows strong signals (blue=positive density; yellow=negative density) on the retinal molecule (red) demonstrating the early isomerization. Surrounding the retinal, changes occur at the amino acid level in an anisotropic direction towards the extracellular side (grey arrow of panel a) along TM5 and TM6 (panels f and g). This anisotropic breathing motion can be detected in the extracellular part of TM3 (e), TM5 (f) and TM6 (g). After 10 ps (panel h) and 100 ps (panel i) of photoactivation, most of the conformational concerted motion changes are reset (h, i and Extended Data Table 3), only a few amino acids only will not revert –like the disulphide bridge C110-C187 (pink arrow)- and take part to further changes along the photoactivation pathway (Extended Data Table 3). Interestingly, we also observe localized intrinsic fluctuations in molecular dynamics simulations of rhodopsin in the dark state (PDBid: 1GZM) analysed from the GPCRmd database⁹⁸. These fluctuations localize at the extracellular side of the transmembrane bundle and are compatible with the energy dissipation changes observed at 1 ps (compare panels c and d (molecular dynamics simulations) with the panels a and b (breathing motion)). Three independent molecular dynamics simulations (3 x 2500 frames) of rhodopsin with retinal (PDBid: 1GZM) were analysed and the backbone root-mean-square fluctuation (RMSF) was depicted on each residue with increasing values from white to red (the RMSF scale was truncated at 1.8 Å for clarity).