This is illustrated by the divergence of the PMF for the bound vanish stage for B2-o (Figure ). The divergence shows a strong correlation with the orientational sampling at λ = 0.325, and not with the presence of water in the binding site (Figure ). For run 4, the plots of ϕ C show that sampling was largely restricted to the orientation of the original binding mode. This resulted in the most positive gradient of the PMF and the most favourable free energy of binding. During runs 1, 2, and 5, the ligand rotated length-ways in the binding site by around 90 degrees with respect to the initial pose, resulting in a less positive PMF gradient. During run 3, the ligand rotated around 180 degrees length-ways in the binding site with respect to the initial pose. The gradient here was evidently even less positive, resulting in a substantially less negative free energy of binding. The slow interconversion between orientations explains the lack of drift of the results with increasing simulation time -there are large barriers between orientations which prevent equilibrium sampling on the timescale of the simulations. To support this explanation, the free energy of releasing B1 to B1-o in the decoupled state was calculated by scaling the strength of the orientational ). This confirms that sampling a large increase in configurational space is not problematic when there are no barriers, at least when the growth is sufficiently slow -the issue is sampling the "rugged" configurational space at intermediate stages of decoupling.