Monitoring selected distances across the active site along our enhanced sampling simulations, we revealed a rich conformational landscape in the active site, with multiple basins corresponding to various orientations of the active site Glu96 and distinct distances between the backbone phosphate A118 and the Mg 2+ ion (Mg 2+ A ) coordinating the attacking water molecule (see Figure ). The center of the basin corresponding to our starting structure is associated with long O2P-Mg A and OE1(Glu96)-P(A117) distances (∼ 6.5 -7 Å), which means that both pairs of groups are separated by more than a single water molecule. However, we identify secondary conformations on the side of this broad basin, with a free energy only 2.0 kcal/mol higher, with Glu96 OE1 oxygen much closer (< 6.0 Å) to the phosphate, which reflects the presence of a single molecule in between those two groups (see Figure ).