TI simulations. As shown in Figure , the relative binding free energy (DDG) can be calculated as the difference between the free energies (DGs) of mutating one ligand to the other in the protein matrix and in solution. Therefore, TI simulations for both process 1 and 2 were performed. For each process, a one-step protocol was adopted, i.e. disappearing one ligand and appearing the other ligand simultaneously. The detailed TI simulation protocol is as follows: First, using the dual topology parameter file, 50000 steps of steepest descent minimization was performed, followed by 1 ns NVT where the system was gradually heated up from 0 K to 300 K which was then followed by 1 ns NPT equilibration at 300 K and 1 bar. These simulations were performed at λ=0.5 to equilibrate the system . No restraint was applied for these simulations and all structures were visually checked. For some mutations, multiple runs had to be performed in order to obtain a stable starting structure. Afterwards the equilibrated structure was used as the starting structure for 12 l windows. For each l, 1 ns of NVT equilibration was performed followed by a 5 ns NVT simulation from which dU/dl data were collected. The efficient 12-point Gaussian quadrature was used for the numerical integration of ∂U/∂λ to obtain all necessary DG values.