After generating the parameters for the individual ligands of a ligand pair, we generated the hybrid topology and structure of the ligand pair using the PMX software package. We concatenated the structure of the hybrid ligand to the structure of its respective protein taken from the complex that contained the trapped water molecule, as our calculations require the position of the trapped water to be known a priori. We solvated the structure of the protein with the hybrid ligand in a box of TIP3P water model 53 and added 0.2 molar Na + and Cl -to the simulation box.