Equilibrium MD simulations in both active and inactive conformations were carried out for a duration of 1 µs each. The following features were computed: RR-hinge-CR inter-domain angle, hinge RMSD to the inactive conformation, the PBC RMSD to the inactive conformation (Figure , Figure ). The domain angle for the inactive conformation remained below 25 o , and in the region of 125-150 o for the active conformation. The hinge RMSD values were similarly well separated and stable, however PBC RMSD showed some overlap between the active and inactive conformations. This occurs because the change in PBC conformation is very small between active and inactive EPAC1 states. These features were used to subsequently build MSMs. These results were sufficient to describe the active and inactive states of EPAC1, by using feature values observed during the equilibrium MD simulations. As the intermediate conformation is defined as a mix of hinge "out" (or inactive-like) and PBC "in" (or activelike) conformations, the definition of the hinge and PBC coordinates was also straightforward.