√ t λ σ ∂H ∂λ λ is dependent on the statistical inefficiency, which is determined by the slowest timescales of exchange between conformational minima which produce different gradients. As a result, it is noisy. Despite the noise, some trends can be observed: √ t λ σ ∂H ∂λ λ is generally highest between λ = 0 and 0.6 for the bound vanish stages where the ligand is more strongly interacting with the protein environment, but subsequently drops as the ligand becomes weakly interacting and sampling is improved. For the bound restrain stage, there are often peaks at λ = 0 where the restraint is absent and the ligand can explore poses which were not observed during the restraint-fitting simulations.