Whilst our simulations on the KE07s were enhanced through Hamiltonian replica exchange simulations, our simulations of PTP1B and the PDZ3 domain were performed using conventional MD and were multi-replica, with 10 independent replicas performed from each state ("closed" and "open" for PTP1B and "bound" and "unliganded" for the PDZ3 domain). This enabled us to evaluate how reproducible using differing numbers of replicas would have been for our analyses of PTP1B and the PDZ3 Domain, and therefore the minimum number of replicas needed to obtain reproducible results. This is important to consider as it has been previously demonstrated that false positive (or false negative) conclusions can be drawn from analyzing a single or only a few replica(s) of conventional MD simulations. To do this, we turned to the statistical techniques bootstrapping with replacement and random subsampling. These techniques are highly similar and involve generating many resamples of a dataset through randomly selecting combinations of observations (with it also possible to duplicate individual observations). applied this method to our KIF calculations on PTP1B and the PDZ3 domain to evaluate how many MD simulations replicas would have been needed to obtain reproducible results. For several different numbers of replicas, we generated 5000 resamples and determined the per residue scores for each resample. We then calculated the correlation between the per residue scores obtained for all 20 unique replicas against each samples' per residue scores, using this to generate KDEs of the distribution of correlation scores possible for a given number of replicas. Results for this analysis using the Spearman's rank correlation coefficient (which measures the ability to rank order the per residue scores correctly) are presented in Figure , whilst those using the Pearson correlation coefficient (which measures the linear correlation) are presented in Figure . We note that an even number of simulations of each state were used for all bootstrap resamples. For example, in the scenario for PTP1B with 10 replicas, 5 replicas each would be obtained from both the "closed" and "open" WPD-loop conformations replicas, see the Methodology section for further details. (B) Bootstrapping with replacement and random subsampling using a random selection of 10 replicas for each bootstrap sample with the time step between each frame used in the analysis altered (meaning those with larger time steps also have correspondingly less frames available). For both PTP1B and the PDZ3 domain, an equal number of simulations from both conformational states ("closed" and "open" WPD-loop or "peptide bound" and "unliganded" respectively) were used in the bootstrapping/subsampling calculations, see the Methodology section for further details. Equivalent results using the Pearson correlation coefficient are provided in Figure .