To ensure that, when the system was in the active state, these stabilizing interactions were possible, only the active state sub-ensemble was recreated using the MSM probabilities. The active state sub-ensemble of EPAC1 L273W shows that the stabilizing hydrogen bonds are indeed prevalent in these conformations (Supplementary Figure ). This confirms that the L273W mutation does not change the cAMP binding pose, but instead changes the hinge-PBC interactions, preventing activation by stabilizing the inactive hinge conformation.