The difference between these two substrate conformations is effectively a 180 o substrate flip, and it is not implausible that both conformations are to some extent catalytic. To test this possibility, we performed empirical valence bond (EVB) simulations of each of the GNCA4-WT, GNCA4-12, V4 (GNCA4-12 with the polypeptide) and V4-4 variants considering both "IN" and "OUT" conformations of the substrate (Figure ). These calculations were performed using both