site. Running absolute hydration free energy calculations of charged ligands in the solvent would have led to a change in the net charge of the system (which is difficult to treat for technical reasons ), while the net charge can be preserved using a relative approach. The ligands were restrained to be half the box edge away from each other using a single harmonic distance restraint (see Section 4.2). We had first attempted restraining ligands to be on top of each other using a single harmonic distance restraint, however, we found that this led to slow convergence of the free energy estimate in some cases. We therefore decided to restrain ligands to be far apart.