with time (Figure ) towards more negative values, which may be due to the requirement for the ligand to sample all points on the surface of a sphere surrounding the protein anchor point upon decoupling. This may be improved by releasing to a centre-of-mass restraint centred on the binding site, reducing the volume which must be sampled. In addition, scaling the restraint potential differently between the end points may improve convergence; instead of the λ 5 scaling used here, a soft bond stretch potential would likely perform well for the removal of these harmonic restraints. This process was repeated using a significantly more permissive multiple distance restraint scheme, where 4 flat-bottomed distance restraints were selected to mimic the 4 protein-ligand hydrogen bonds shown in Figure . This scheme is denoted M-Hand-R as the anchor points were selected by hand, although automated selection to match hydrogen bonds would be straightforward. The radius of the flat-bottomed region was selected to be as small as possible without the restraints engaging at any point during the restraint fitting simulation, ensuring that ∆G Bound, Restrain ≈ 0. The force constants for the half-harmonic potentials were 40 kcal mol -1 . This again produced a ∆G o Bound in good agreement with the Boresch results and M-Rig (-6.47 ± 1.16 kcal mol -1 ), indicating that the relatively permissive restraints sufficiently restricted orientational sampling. Furthermore, no substantial drift in the estimate with simulation time was observed (Figure ).