These free energy simulations used the same force field parameters and core simulation settings (e.g., time step, temperature, pressure, treatment of short-and long-ranged nonbonded interactions) as the MD equilibration stage described above. Each window ran for 1 to 3ns of simulated time, with about two thirds of the MD time corresponding to production stage simulations whose frames enter the free energy analysis. Further details of the BAT.py procedures can be found in a previous publication and on the BAT.py Github page. Using RTX3090 GPU yielding ∼300 ns/day of simulated time for the solvated systems in this study, the free energy simulations can be completed in about 2 hours wall time when running in parallel with dozens of GPU, or in about 1 day per pose when running sequentially with single GPU. This free energy stage is the computational bottleneck in the overall procedure, because the MD equilibration stage and docking are much cheaper computationally.