Finally, RNase H structures 18 suggest that the nucleophilic water molecule, coordinated to the Mg 2+ A ion, could donate a H-bond to the downstream phosphate. This feature led computational studies to consider the downstream phosphate as a potential general base for the initial proton transfer. The starting structure of the M5:rRNA complex does not exhibit such a feature, with a downstream phosphate (A118) too far from the active site to allow formation of a single-water bridge. However, our enhanced sampling simulations revealed the conformational flexibility of the active site, and the existence of an alternative conformation where the A118 phosphate group has moved close enough to be bridged to one of the active site cations by a single water molecule, similarly to what was observed in RNase H. Our simulations further suggest that the two conformations could have similar populations. While not explicitly discussed and quantified in the case of RNase H, such flexibility of the active site may be also relevant there, as earlier studies 14 mention that the interaction between the nucleophilic water molecule and downstream phosphate may be either direct or mediated by another water molecule.