RBFE PLpro system setup and TI simulations. Four ligands were selected from our previous work on the PLpro system and are displayed below in Figure . Ligands 1-3 were selected such that their ∆∆𝐺 𝐴→𝐵 𝑏𝑖𝑛𝑑 calculated in previous work were negative (Ligand 1), approximately 0 kcal/mol (Ligand 2), and positive (Ligand 3). Ligand 4 was selected as an edge case due to the difficulty of the mutation (see Discussion). Input coordinates, topologies, and parameters were obtained from our previous work (see for details). All λ-windows were minimized and equilibrated using the protocol described in the previous section, with restraints applied to two water molecules in the binding pocket during minimization and heating, and gradually removed during density equilibration. For each mutation, 10 replicates were performed per protocol. All uncertainties were quantified using samples of multiple replicates.