To obtain equilibrium distributions of the coupled protein-ligand and protein-only systems at 298 K, harmonic position restraints with a force constant of 9000 kJ/mol/nm 2 were applied to all protein and ligand atoms of the above initial structures and the energy was minimized followed by a 300 ps simulation in the NVT ensemble, where the first 5 ps were used to bring the temperature of the system from 0 to 298 K with simulated annealing. Position restraints were then relaxed to 500 kJ/mol/nm 2 for a 50 ps NVT simulation followed by a 10 ns production NPT simulation without any position restraints. For the leg of the thermodynamic cycle of ligands in water, the simulations used no position restraints. Firstly, energy minimization was performed followed by the 10 ps NVT and 10 ns production NPT simulations.