We note that the selected starting structure for the mechanism is not in the center of the main population basin, as Glu96 is closer to the scissile phosphate than in the dominant geometry (see Figure ), which allows the nucleophilic water molecule to be directly deprotonated by Glu96. In the dominant conformation of the active site, direct proton transfer is not possible, but could instead happen through a string of two water molecules (Figure ). Characterization of the free energy profile associated with such a mechanism however shows that it is associated with a much higher free energy barrier (see Table and Figures S12, S13, S14). These results thus suggest that from the dominant conformation of the active site, the most favorable route would be first a conformational fluctuation to bring Glu96 in an appropriate geometry (with an estimated free energy cost of about 2 kcal mol -1 ), followed by direct deprotonation of the nucleophilic water molecule by Glu96 from this specific geometry.