For each system, the endpoints of the ten equilibration runs were then used as starting structures for subsequent EVB simulations, with three additional equilibration runs of 500 ps in length being performed from each of these starting points, using new random velocities, in order to generate 30 discrete starting points for EVB simulations of each system. The EVB free energy perturbation/umbrella sampling (EVB-FEP/US) calculations were performed in 51 individual mapping frames of 100 ps simulation length each, leading to a total of 5.1 ns simulation time per individual EVB trajectory, 153 ns simulation time per system, and 4.590 μs of equilibration time over all systems considered in this work. The EVB parameters were calibrated using the uncatalyzed background reaction in aqueous solution as a baseline, as described in ref. . The same calibration as in our previous work was used in the present study, and no new calibration was performed here with all EVB parameters used in this work presented in the Supplementary Information of ref. .