However, these calculations still allow us to estimate which of the two substrate conformations are energetically preferred, as we are averaging over 30 independent trajectories (with conformational sampling across the trajectory) per system. Based on these calculations, no significant differences are observed in the free energies calculated for "IN" and "OUT" conformations for each of the systems when comparing the different variants to each other (direct comparison of the same substrate binding across the variants). However, for all the variants the energy of the "OUT" conformation is larger than the "IN" one, indicating that the "IN" conformation will favor the enzymatic reaction in all variants. This is also the conformation that is expected to be catalytically productive based on the positioning of the substrate inside the de novo active site. In addition, it is not implausible (see the Supplementary Discussion) that the "IN" conformation promotes catalysis through a higher value of the pKa of the catalytic group, a localenvironment effect that could perhaps be more pronounced in the more active Kemp eliminases.