Unless otherwise mentioned, a hydrogen mass repartitioning scheme (HMR) was used to achieve a 4 fs integration timestep for our simulations. Due to differences in how MD engines handle hydrogens with analytical constraints , hydrogen masses, except those of waters, were increased to 3 and 4 atomic mass units for AMBER and GROMACS respectively. Whilst HMR has been employed before in alchemical free energy calculations , there is limited data on its use in absolute binding free energies in GROMACS. To this end, a small validation using the Cyclophilin D dataset is shown in Supplementary Information (see SI Text S3).