Figure 5.

Metadynamics simulations of tRNA acylated with α- and β²-hydroxy and amino acids prior to bond formation in a reduced ribosome model (RRM). (A) Free energy surface (FES) plots of 30 Å RRM containing a P-site tRNA^Met acylated with fMet and A-site tRNA^Met acylated with (S)-α-NH₂1, (S)-α-OH 2, (S)-β²-OH 3, or (R)-β²-OH 4, plotted along the collective variables of Bürgi–Dunitz angle α_BD and N_α–C_sp2 distance. 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 (Supporting Information Figure 10). The color scale represents the free energy in kilocalories per mole (kcal/mol), 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. Average N_α–C_sp2 distances and α_BD angles for the 0–1 kcal/mol energy minima of each plot are displayed below (gray boxes). (B) Representative conformation and relative geometry of the P-site (gray) and A-site (colored) monomers from the frames at the free energy minimum of each FES plot. Poses were chosen to highlight the N_α–C_sp2 distance (black dotted line) and the Bürgi–Dunitz angle α_BD 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. (C) Pairwise interaction difference analysis (PIA) plots displaying the pairwise-distance differences between the C1′ carbons of nucleotides within 5 Å of each A-site monomer ((S)-α-NH₂1, (S)-α-OH 2, (S)-β²-OH 3, or (R)-β²-OH 4) along the metadynamics trajectories. Heatmaps were constructed by subtracting the C1′ distances within the RRM containing tRNA charged with the indicated monomer from the C1′ distances within the analogous RRM containing (S)-α-NH₂1. Darker red indicates greater distances between nucleotides for RRMs containing the indicated monomer ((S)-α-OH 2, (S)-β²-OH 3, or (R)-β²-OH 4) relative to that containing (S)-α-NH₂1, whereas darker blue indicates smaller distances.