The stationary points corresponding to stable and metastable states along the FE surfaces (local energy minima) obtained from WTMD simulations have been characterized by calculating the average values of the structural descriptors of the system. To compute average values from equilibrium distributions, representative structures correlating with minima on the WTMD FE curves were extracted and subjected to 50 ps-long unbiased QM/MM MD simulations adopting the same setup described beforehand for the unbiased simulations. The fluctuations of the features during these unbiased trajectories were monitored with PLUMED 2.9, 61 and they include the distances and coordination numbers used to optimize the reaction coordinate via HLDA (d1-d11 and c1-c3). Moreover, two further distances (d12 and d13) and two angles (a1 and a2) have been analyzed to assess the stabilization of the carbonyl O5'' atom by the oxyanion hole. The distances d12 and d13 were calculated between the O5'' atom of PET4 or PEF4 and the amide H atom of the M161 and Y87 backbones, respectively, while a1 and a2 correspond to the N-H---O angle formed by the amide N and Hatoms of the M161 and Y87 backbones and the O5'' atom of PET4 or PEF4 (Figure ).