In contrast to the standard deviations, the time-normalised standard errors of the means of the gradients converge slowly (Figure ). Increasing sampling time produces higher √ t λ σ ∂H ∂λ λ values which again reflects the fact that the uncertainties do not generally decrease with sampling time. This increase can happen for two reasons; firstly, because the replicates remain trapped in local minima in configuration space , producing constant σ ∂H ∂λ λ at increased time and hence increased √ t λ σ ∂H ∂λ λ . In other words, our estimate of the statistical inefficiency has failed to converge because no transitions between the relevant conformational minima have been observed. Alternatively, this can be due to replicates exploring new local minima in configuration space, increasing both σ ∂H ∂λ λ and √ t λ σ ∂H ∂λ λ . As expected from the uncertainties in Table , √ t λ σ ∂H ∂λ λ is generally much smaller and more consistent with increasing simulation times for the free legs over the bound legs, reflecting the fact that the protein environment poses substantial sampling challenges which dramatically increase the statistical inefficiency. The exception to this is the free vanish stage for Nutlin, which shows a substantial peak in √ t λ σ ∂H ∂λ λ around λ = 0.5 which is similar in magnitude between the bound and free legs. This is due to the overlap of intramolecular groups (Section S9).