In the case of PTP1B, even a single pair of replicas (1 from the "closed" conformation and 1 from the "open" conformation) shows quite reproducible results according to both correlation metrics used (Figures and). This contrasts strongly with the PDZ3 domain whereby highly misleading results could be obtained from just a single pair of replicas (Figures and). This contrast is likely a result of two factors, the first being that magnitude of the difference in the conformational states of PTP1B is larger than that of the PDZ3 domain. That is, whilst in PTP1B the largest differences will be from interactions present in one state and not present at all in the other, for the PDZ3 domain, interactions will generally be present in both states but stronger in one state, therefore requiring more sampling to reliably differentiate. The second is the available crystal structures of PTP1B in both the closed and open states, enabling us to confidently assign and simulate both states. In contrast, simulations of the PDZ3 domain's unliganded state were performed using the peptide bound crystal structure (after a 100 ns equilibration simulation, see the Methodology section). Another consideration is that the sampling of each state was obtained using different simulations, for example, the PDZ3 domain peptide bound simulations were used to obtain frames for the peptide bound state and vice versa for the unliganded state. For simulations in which both (or more) states are obtained from the same set simulations (i.e., sampling back and forth between states in each replica), the sampling required would also likely be affected by the decorrelation times associated with transitioning from one state to another. That is, the time taken for the structure to fully adapt to the new conformation/configuration after undergoing a conformational change away from another conformation.