During MM MD, the carbapenem 6α-hydroxyethyl group was able to rotate to occupy three different orientations, which can be distinguished by the value of the C7-C6-C-O dihedral angle: around 50°, 180°, or 290°, henceforth referred to as orientations I, II, and III, respectively (Figure ). The 6α-hydroxyethyl orientation affects interactions in the active site, because its hydroxyl group can hydrogen bond either with the DW (I), or with the Lys73 carboxylate (III), or stay close to the crystallographically observed pose, in which its methyl group is positioned next to the DW and points towards Leu158 (II, Figure ). The starting orientation of the 6α-hydroxyethyl for both carbapenems is II, as in the crystal structures used in model construction. During MD si mulations, this sidechain is free to move and sample all three orientations. For meropenem, orientation I is sampled more than II, while III is sampled only minimally (Figure ). Conversely, both orientations II and III are sampled more than I for imipenem. The free energy difference between the different orientations of the 6α-hydroxyethyl group was estimated by calculating the ratio of MD trajectory frames corresponding to each orientation (Z), and using ΔG=RTln(Z), where R is the molar gas constant and T the simulation temperature (300 K). For imipenem, the lowest free energy state is orientation II, with slightly higher relative energies of 0.6 and 0.2 kcal/mol for orientations I and III, respectively. For meropenem, orientation I has the lowest free energy, orientation II is slightly higher (0.6 kcal/mol) but orientation III is significantly higher (2.2 kcal/mol). The presence of a methyl group in the 1β-position in meropenem (instead of a 1β-proton in imipenem) may explain the relatively higher penalty for orientation III, as in this orientation the 1β-substituent is located directly next to the 6α-hydroxyethyl moiety.