All solvated systems (both "initial" and obtained from Alibay et al.) were subject to the standard A3FE minimisation and equilibration workflow, in which all simulations were performed using GROMACS 2021.3 through BioSimSpace (version 2023.0.0). Systems were energy minimised for 1000 steps, followed by equilibration in the N V T ensemble (5 ps with all non-solvent atoms restrained and heating from 0 to 298 K, followed by 50 ps with restraints on all backbone atoms for the complexes only, then 50 ps with no restraints). N P T equilibration was then performed at 1 atm and 298 K (400 ps with restraints on non-solvent heavy atoms, followed by 1 ns with no restraints). Finally, independent 5 ns N P T equilibration runs were carried out for each independent replicate run for all systems to provide varied starting conformations. All restraints used a force constant of 10 kcal mol -1 Å-2 . The V-rescale and C-rescale algorithms were used for the relevant equilibration steps, with time constants of 1 and 4 ps, respectively. according to the thermodynamic cycle shown in Figure of Clark et al.. The simulations with the ligand in solvent collectively make up the free leg, while those with the receptorligand complex make up the bound leg. Sets of calculations where interactions of a given type are introduced or removed are termed stages: receptor-ligand restraints were introduced, charges were scaled, and Lennard-Jones (LJ) terms were scaled in the restrain, discharge, and vanish stages, respectively. Standard free energies of binding were calculated according to Bonds to hydrogen were constrained. A cut-off of 12 Å was used for all non-bonded interactions and electrostatics were treated with the reaction field method with a dielectric constant of 78.3. We did not correct for the truncation of the tails of the LJ potentials because we found this produced negligible corrections. Energy minimisation with a maximum of 1000 iterations was performed prior to every simulation. The gradient of the free energy with respect to λ was calculated every 200 timesteps.