Although the aim of this study was to compare the values of ∆G o Bound obtained with different restraints, a single set of five replicate calculations were carried out for the free leg to allow comparison with experiment. Comparison with experiment cannot be used for the comparison of restraint schemes as there are likely to be systematic errors from the force field, but it is used here as a crude check on the overall results. There was excellent agreement between replicates of the free leg simulations and convergence was achieved quickly (Section S10), yielding ∆G Free = -3.08 ± 0.14 kcal mol -1 (∆G Free, Discharge = 9.03 ± 0.07 kcal mol -1 , and ∆G Free, Vanish = -12.11 ± 0.11 kcal mol -1 ). This was in spite of syn-anti interconversion, which was observed during at least one run for every lambda window, but which occurred slowly on the timescale of the simulations. ∆G o Bound for binding pose B (with the B1-poseB restraints) was -6.63 ± 1.11 kcal mol The lack of orientational restraints allows the mixing of binding poses A and B, but this is also not a plausible source of the error introduced. In the limit of perfect sampling, the free energy of binding can be no more negative than that calculated by combining the free energies of binding of the two poses as was done previously. The exception to this would be if there were other binding poses which were numerous or more favourable, which seems unlikely.