Lag-times were between 25-100 ns whilst the number of metastable states chosen were between 4 and 7, see Table . These parameters were validated by a Chapman-Kolmogorov test for each system (Figures ). Metastable states were identified using the perron-cluster cluster analysis (PCCA+) were then used to determine the mean first passage times (MFPTs) for each system as implemented in PyEMMA. The free energy differences were obtained from the MFPTs by calculating the equilibrium constants from the forward and backward rates (and, from that, ΔG).