The potential energies for simulations of the corresponding protein-ligand complexes are given analogously as U P L,aX . Equations 1 and 2 report the energetic consequences of going from the flexible to the covalently constrained ligands, while Equations 3 and 4 report the energetic consequences of going from a central valine residue to a glutamine. In order to obtain correctly balanced energies, the numbers and protonation states of waters, ions and buffer compounds were identical across all simulations, except for the addition of counterions required to maintain electrical neutrality; see Section 3.2.