Representative structures from our simulations of the GNCA4-WT β-lactamase are shown in Figure , with average donor-acceptor distances from our simulations highlighted. The corresponding donor-acceptor distances and donor-hydrogen-acceptor angles for all variants shown in Table can be found in Table . Finally, the electrostatic contributions of individual residues to the calculated activation free energies can be found in Figure . These contributions were calculated by applying the linear response approximation (LRA) to our calculated EVB trajectories, as in our previous work (e.g. refs. ). From this data, it can be seen that the Figure and Table show an overview of the calculated activation free energies for the top 20 FuncLib variants. From this data, it can be seen that in the majority of variants, we obtain very little differences in activation free energy (similar to the prior results shown in Table ), with at most 1 kcal•mol -1 improvement compared to GNCA4-WT. The only exception to this is a variant (GNCA4-4) with a high activation free energy of 20.3 kcal•mol -1 . This is due to the introduction of an I250M substitution in this variant. Here, the longer side chain of methionine is located between the substrate and the catalytic D229 side chain, introducing steric hindrance in the active site that displaces the substrate from an optimal binding position and increases the D…A distance at the Michaelis complex substantially (see Table ). All other calculated values based on FuncLib predicted structures lie in the range of 15.3 -17.4 kcal•mol -1 , compared to a calculation activation free energy of 16.2 kcal•mol -1 for the wild-type enzyme (Table ). , as well as the corresponding calculated activation free energies based on either structures predicted from FuncLib (∆G ‡ calc,FL) or, where available, directly from crystal structures (∆G ‡ calc,XTL). All energies are presented in kcal•mol -1 , and the calculated activation free energies are averages and standard error of the mean over 30 individual EVB trajectories per system, as described in the Materials and Methods. The raw data for this figure can be found in Table .