Titrations were performed over 20 B values loosely centered around the experimental binding free energy, although knowledge of the experimental binding affinity is not necessary and the titration could be performed over any B range. B values were calculated using Equation 3 with a fixed µ ′ sol value for each ligand calculated using a basic hydration free energy calculation as described above. Insertions and deletions were performed with a switching time of 150 ps. In each cycle, a GCNCMC move was attempted for every 1 ps of MD. 1700 cycles were performed, with the first 200 discarded as equilibration, giving a maximal production time of 76.5 ns per B value.