Fig. 5: Plots of FESs from metadynamics simulations.
FESs of 30 Å RRM containing a P-site tRNA acylated with fMet and A-site tRNAs acylated with monomers 1–8, plotted along the collective variables of Bürgi–Dunitz angle αBD and Nα–Csp2 distance. The metadynamics simulations that generated each FES began with the 2.1 Å cryo-EM model reported here, but the A-site Met was in silico mutated to the other monomers, while the P-site Met was converted to fMet. Each FES shown is the average of two metadynamics runs starting from orientations of the A-site monomers that differ by a 180° rotation about the ψ angle. The colour scale represents the free energy in kilocalories per mole, where the global minima are set at 0 and therefore the various heights of the energy scales are based on the energetics of the fluctuations of the A-site monomers. The conformation and relative geometry of the P-site (rose) and A-site (green) monomers at the free energy minimum are shown above each plot. Poses were chosen to highlight the Nα–Csp2 distance and the Bürgi–Dunitz angle αBD at the free energy minimum. The tRNAs to which each monomer is attached as well as the RRM surrounding the two monomers have been omitted for clarity. Coordinates for the lowest energy models obtained for each simulation are available at https://doi.org/10.5281/zenodo.7730661. Two methods were used to establish that convergence was reached during the 100 ns runs. First, trajectory plots representing the Nα–Csp2 distance and Bürgi–Dunitz angle (αBD) for metadynamics replicas reveal that the conformation space is revisited multiple times over the course of the metadynamics runs (Supplementary Figs. 2 and 3). Second, FESs calculated for the last 80 ns of the 100 ns simulation are qualitatively similar to those calculated after 100 ns, with the differences between the FESs of the last 80 ns and after 100 ns on average ~0.8 kcal mol–1 (Supplementary Figs. 4–6).
