Each ABFE window consists of a short equilibration similar in protocol to the previously detailed equilibration cycles, although using 10 ps for the restrained NVT equilibration, 100 ps for the restrained NPT Berendsen barostat equilibration, and 500 ps for the unrestrained NPT Parinnello-Rahman barostat equilibration. This is followed by 20 ns of production NPT simulation. A total of five independent replicas of the ABFE cycle are simulated. For the protein-ligand complexes, each replica is independently equilibrated and uses different orientational restraints, allowing the replicas to more representatively sample the available conformational space within the binding site. GROMACS 2021 was used for all ABFE calculations. For charged ligands, to reproduce the work of Rocklin et al.