To sample the active site conformations of the two KE07 variants described above, we performed eight Hamiltonian replica exchange MD (HREX-MD) simulations of 200 ns length each, using the same protocol as described in our prior work. HREX-MD is a form of enhanced sampling simulation and with these simulations we were able to generate free energy landscapes representing the relative stability of the two major active site conformations observed in the crystal structures of these variants using the χ1 and χ2 angles οf W50 (Figure ). We have selected the R4 variant, in particular, as this is the first variant in which a sizeable population of the catalytically important C conformation is observed. Note that for conciseness, we do not discuss here the conformational flip of the E101 side chain shown in Figure , for further discussion of this shift see ref. . shows a population shift towards the "C" state and away from the "A" state from R1 to R4, in line with prior work. Note that, as pointed out in prior work, the "B" state appears to be a transient conformation that appears in the transition from the "A" and "C" states. Confident that our simulations could reproduce the previously observed population shift, we applied our package to help understand how this population shift had occurred through the 4 mutations that separate R1 and R4 (I7D, K146E, G202R, and N224D).