Initial structures for the 14 proteins were taken from downloaded PDB datasets and aligned to those from previous FEP+ studies. For ligands, they were derived from the supplementary data provided with prior studies. All crystal water molecules within 5 Å of the protein were included. Protonation states and tautomeric forms for histidine residues were kept consistent with previous FEP+ studies. GAFF (v2) parameters were used to prepare ligand molecules with charges calculated using AM1-BCC model. The AMBER ff14SBonlysc forcefield was used to parameterise proteins. Our systems were solvated in an orthorhombic TIP3P water box with at least 14 Å solvent in all directions. Sodium and chloride ions were used to neutralise the system electrostatically employing Joung-Cheatham ion parameters. 65 AmberTools20 66 was used to perform parameterisations and prepare all models.