All simulations were performed using NAMD . For all equilibrium simulations (that is, those with fixed λ values in both the EQ and the NEQ approach), the ligand in the protein environment was first energy minimised, followed by a 20 ps NVT equilibration and a 2 ns NPT equilibration. Pressure and temperature were maintained at 1 atm and 300 K using a Berendsen barostat (compressibility of 4.57 × 10 -5 bar -1 and relaxation time of 100 fs) and a Langevin thermostat (damping coefficient of 5 ps -1 ), respectively. The production run time was taken to be 10 ns after a systematic and extensive analysis (as detailed in the Results section). A time step of 2 fs was used. When a ligand in the water environment was simulated, the entire protocol remained the same except that the NPT equilibration step was only 1 ns long. During both minimisation and equilibration steps, protein backbone atoms were initially constrained to their initial positions and were slowly allowed to relax. Periodic boundary conditions were employed with long-range electrostatics handled by the Particle Mesh Ewald (PME) method . A nonbonded cutoff of 12 Å was used. The van der Waals interactions were smoothly switched off between 10 and 12 Å, being linearly perturbed between λ value 0 and 1. The standard NAMD soft-core potential was used for the van der Waals terms with the radius-shifting coefficient of 5 Å 2 to avoid singularities. While the coordinates were recorded every 10 ps, energy derivatives with respect to λ were recorded every 2 ps.