However, the appropriate domain angle values to be used for the definition of the intermediate state were not evident from the equilibrium MD simulations, as EPAC1 remained in its active or inactive starting conformation on a 1 µs timescale. To choose a suitable domain angle value for the intermediate state, a simulation steering EPAC1 from inactive to active conformation (outlined below) was aligned to an EPAC2 structure with RAP bound (PDB ID 3CF6). Only the catalytic regions were used for alignment, to capture the translation of the regulatory region. Pairwise C(α)-C(α) distances between all EPAC1 RR and all RAP residues were computed for the sMD simulation, taking the smallest one as the EPAC1(RR)-RAP distance. This distance was plotted against the domain angle, to provide a direct relation between the inter-domain angle and the occlusion of the RAP binding site by the cNBD (Figure ). While the domain angle increased from 20 o to 60 o , the minimum Cα-Cα distance remained low, but jumped up as the domain angle increased from 60 o to 95 o (Supplementary Figure ). Therefore, the inter-domain angle of 60 o was chosen to define the centre of the intermediate state, with values of 30 o and 100 o chosen for the centres of the inactive and active states respectively.