The sMD/MSM workflow shown in Figure was applied to apo EPAC1 and the EPAC1-cAMP complex. Example sMD results are shown in Supplementary Figure . MSMs for each system were built with a 25 ns lag time, based on implied timescales (ITS) shown in Supplementary Figure . The equilibrium probability of each microstate was computed from the resulting MSMs. The probability density of each state is shown in Figure , along with the macrostate partitioning is plotted as a function of the hinge and PBC RMSD features. In agreement with experimental results, apo EPAC1 is modelled as primarily adopting the inactive conformation (63%, Inter Quartile Range (IQR) 55-70%), some intermediate conformation (37%, IQR 30-44%), and no active conformation (0%, IQR 0-0%) (Figure ). These values are reflected in the stationary probability distribution shown in Figure , where the majority of the density is seen at low hinge and PBC RMSD values, and no density is seen in the high value region. In the presence of cAMP, the active state probability increases to 91% (IQR 81-100%), demonstrating that the sMD/MSM approach applied here has captured strong activation by cAMP. Additionally, the cAMP stationary probability density in Figure shows an increase in probability density in the high regions of hinge and PBC RMSD values, corresponding to the active state.