Overall, there is (from a computational perspective) good agreement with the experimental values, with the calculated values falling to within 2 kcal•mol -1 of experiment, considering that unlike the calculations on the simpler single amino acid substitutions shown in Figure , in the case of the FuncLib variants, we are now making predictions for the effect of multiple simultaneous variants using computationally predicted structures. Our data is also in agreement with other computational studies of Kemp elimination, that report activation free energies within 2 (or sometimes more) kcal•mol -1 from experiment. We note that we have attempted to further refine our EVB calculations by exploring other approaches to generate the starting structures, such as predicting mutations using SCWRL or inserting point mutations manually using the Dunbrack rotamer library, as implemented in Chimera. We tried comparing all three approaches using the GNCA4-2, GNCA4-10 and GNCA4-17 variants as model systems, as these variants show some of the greatest deviations from the experimental values (Table ). However, the resulting activation free energies were within 0.4 kcal•mol -1 of the values obtained using the FuncLib structures (Table ), with the exception of GNCA4-2/SCWRL which yielded an activation free energy of 16.0 ± 0.3 kcal•mol -1 in better agreement with the experimental value of 15.5 kcal•mol -1 (note however that in the case of GNCA4-2, there was a TSA bound in the active site to guide substrate placement). Therefore, we did not pursue these avenues further as we did not observe systematic improvement in our calculated activation free energies by using alternate approaches to generate the starting structures.