The program BAT.py version 2.0 was used to prepare the equilibration simulations, which were run with the simulation engine AMBER . This is referred to as the equil stage in BAT.py. The simulations used the exact same prepared protein-ligand system as did the docking calculations, except that crystallographic waters which do not clash with any ligand in the compound set were now included. The protein with its docked ligand was exported from Maestro and parsed into AMBER files to build the simulation system. The AMBER ff14SB force field was used for the protein, GAFF version 1 with AM1/BCC charges was used for the ligand, and the TIP3P model was used for water molecules. Bulk water molecules were added to form a cubic solvent box. The resulting system was energy-minimized, and translational and rotation restraints of the ligand relative to the receptor were applied, using the same scheme and force constants as for the subsequent ABFE calculations (BAT.py user manual). MD simulations, with a time step of 4fs, hydrogen mass repartitioning , temperature control via Langevin dynamics , and Monte Carlo barostat, were then carried out in four stages: heating to 298K over 0.1ns, NPT equilibration over 0.4ns, gradual release of ligand restraints over 0.4ns, and finally a 12ns MD simulation with Monte Carlo/MD exchange of waters near the ligand .