At each stage m, an independent MD simulation was performed. The simulation time for each stage m was 17.5 ns, where the first 1.5 ns was discarded from analysis as equilibration. The change in the Hamiltonian with the current configuration between stage m and the other stages is computed every 0.20 ps. This is saved for subsequent post-simulation analysis with MBAR to determine ∆ G solv 1 . This analysis was performed using the Python implementation of MBAR (PyMBAR) and the GROMACS analysis script distributed with it .