Although not explored in the present work, one might expect similar issues to arise in normal mode analysis based on fixed-atom constraints, as used for vibrational spectroscopy simulations of metalloenzymes. The focus here is on calcula- We next consider a realistic enzyme model and investigate the effect of harmonic restraining potentials on the shape of the potential surface, for a one-dimensional scan along a flexible torsion angle. These calculations employ a cluster model of the active site of threonyl-tRNA synthetase (ThrRS), in which p-biphenylalanine is found with the biphenyl moiety in a coplanar conformation (PDB: 4S03). The charge-neutral cluster model is taken from Ref. 82 and which is shown in Fig. , highlighting the biphenyl moiety. We constructed a onedimensional scan around the indicated dihedral angle (θ) between phenyl rings of the biphenyl moiety, relaxing other degrees of freedom at each fixed value of θ. These one-dimensional scans are plotted in Fig. using either vacuum boundary conditions (ε = 1) or else dielectric boundary conditions with ε = 4, and carried out using either fixed-atom constraints or harmonic confining potentials for the anchor atoms.