We note also that unlike in the case of these geometric parameters (Figure ), we do not observe significant correlations with other energetic features of the reaction such as the pKa of the catalytic base (predicted using PROPKA 3.1 62 ) or the reorganization energies. Therefore, it is likely that a significant component of the calculated changes in activity observed upon introduction of the amino acid substitutions predicted by FuncLib is better geometric preorganization of the active site for efficient proton abstraction from the substrate, as was also observed in the case of the crystal structure of the directed-evolution optimized Kemp eliminase HG3.17, compared to the computationally designed HG3. Finally, one additional feature that can be reducing the quality of our predictions is the fact that the FuncLib variants involve the introduction of up to nine mutations into each structure (of the eleven positions that were selected for randomization, see the Supplementary Data), which is likely to compromise the quality of the FuncLib generated protein structures. To assess this, we also performed EVB simulations on the variants for which crystal structures were available: