The GAFF force field was used for all ligands and the ff99SBildn force field for the protein. For each transformation, topology and coordinate files were generated for the initial and the final conformation. Common atoms in both ligands were checked to have the same initial coordinates, and differing atoms were specified in the scmask. The crgmask contained atoms with the atomic partial charges set to zero. To switch on the free energy calculation, icfe and klambda were set to 1. clambda was set to 5 values between 0 and 1 ( l-value of 0.046, 0.230, 0.5, 0.769, or 0.953, respectively). ifsc was set to 1.