It is also possible that the contribution of R55 to electric fields in CypA was previously overestimated. First, field strengths were previously calculated at the proline N atom only, rather than averaged across both peptide bond atoms, and we consistently see a larger electric field at the proline N than the preceding C atom in all force fields tested in our calculations (Table ). Second, although fields were previously calculated using DFT ONIOM calculations at the B3LYP/6-31G** level, only a subset of the CypA protein (250 atoms) was included in the QM region, and without long-range electrostatic effects of protein and solvent estimated by electrostatic embedding. Moreover, electric fields were calculated taking into account the contribution of only the CypA receptor residues, not using the environment fields approach of Fried & Wang that includes back-polarization of the receptor by the substrate. It is difficult to predict the modulation of electric field caused by back-polarization at a specific site along a specific vector, but the effect should strictly be included as far as practicable. Finally, the previous WT CypA simulations were performed incorporating experimentally-derived restraints, and should therefore be a reliable representation of the WT structural ensembles. However, trajectory frames were geometry optimized using the Amber ff99SB-ILDN force field prior to the DFT calculations, and structures of the R55A mutant were generated directly from the WT structural ensemble rather than a separate set of simulations. The previously reported effects of the R55A mutation are therefore purely electrostatic, and do not consider the potential for any structural reconfiguration upon mutation. Both approximations are conceptually likely to overstabilize and hence overestimate the electrostatic effects within the CypA active site.