Because the interactions of the 6α-hydroxyethyl group in the active site have been suggested to play a role in modulating β-lactamase activity towards carbapenems, deacylation free energy barriers were calculated separately for all three orientations of both imipenem and meropenem acylenzymes observed in MD simulations. Starting structures for US were chosen from the 200 ns MM MD simulations following two criteria: that a potential DW was at a suitable distance for nucleophilic attack, and the 6α-hydroxyethyl orientation was that desired. For orientations II and III, the sidechain dihedral was restrained close to the reference values to avoid the substrate changing between orientations during the reaction (no restraints were needed for I, as no sidechain rotation was observed during US). Overall barriers for deacylation were determined by combining sampling from three separate US calculations for each AC conformation (with different starting structures), with standard deviations calculated between the free energy barriers for individual US simulations (Table ). More details of the US setup and analysis are available in the SI. Calculated deacylation free energy barriers for the ACs formed by imipenem and meropenem with the 6α-hydroxyethyl in each of the three orientations are shown in Figure . For all orientations, two barriers are shown, corresponding to two different hydration states around the general base.