In order to study the torsional free energy associated with rotation of the conserved W-loop tryptophan, we performed eight 400 ns-long well-tempered metadynamics 27 simulations (one per system), using the AMBER 2020 32 simulation package and Plumed 2.7. We used as collective variables the ꭓ1 (N-Cα-Cβ-Cγ) and ꭓ2 (Cα-Cβ-Cγ-Cδ2) dihedral angles, which describe the torsional space of the tryptophan side chain. Metadynamics runs were started after 50 ns of NPT equilibration (following the same protocol and simulation settings as the conventional MD). The backbone root mean square deviation (RMSD) of the helices and β-sheets of the protein structure, and the diffusive behavior of our metadynamics runs, are shown in Figures and, respectively. Simulations were performed using a bias factor of 12, a Gaussian width of 9 degrees per CV, a Gaussian initial height of 0.1 kcal/mol, and a deposition rate of 1 ps -1 . Convergence of these simulations was checked by monitoring the evolution of the CVs over time together with the free energy profile.