ABFE lysozyme TI simulations. For TI simulations of solvated ligand, the ligand was solvated in a TIP3P water box using tleap with a 15 Å distance between the ligand and the edge of the box. For TI simulations of the protein-ligand complex, the orientation of ligand with respect to the protein were restrained using the virtual bond approach (see SI for details regarding the selection of atoms for the virtual bond). Force constants of 4 kcal/(mol*Å 2 ), 20 kcal/(mol*rad 2 ) and 40 kcal/(mol*rad 2 ) were used for distance, angle and dihedral angle restraints, respectively. The second-order smoothstep softcore potential (SSC( )), as implemented in AMBER20, was utilized for both the protein-ligand complex and solvated ligand steps. For each λ-window, the system was minimized and then equilibrated using the same protocol as was performed for conventional MD.