GNCA-2, GNCA-12 and GNCA-19. For these variants, the calculated values fall to within 1.3 kcal•mol -1 of the experimental values, and can deviate by up to 2.2 kcal•mol -1 from the values calculated from the FuncLib predicted structures. In the case of GNCA-2, which has the largest deviation between the calculated activation free energies using the crystal and FuncLib structures (∆∆G ‡ calc = 2.2 kcal•mol -1 with the crystal structure giving better agreement with experiment), we observed subtle structural differences the crystal and FuncLib structures (Figure ). Specifically, we observe different rotamers of the R256 and L291 side chains, as well as also subtle displacements of both the side chain of the catalytic base D229 (which is further from the substrate in the FuncLib predicted structure). The shift in the position of the catalytic base D229 in particular likely plays a significant role in the higher calculated activation free energy for this variant when using the FuncLib predicted structure as a starting point.