It appears that initializing ligand coupling simulations from holo structures leaves the binding site -in particular the T106 rotamerpre-arranged to accommodate the ligand. This, in turn, leads to an overly stabilized proteinligand complex as quantified by the binding ∆G. The missing term in ∆G, in this case, is the free energy required to switch T106 rotamer from its apo to holo state. To demonstrate this, we also computed free energy surfaces for the residue's χ1 dihedral with welltempered metadynamics simulations biasing the potential of the dihedral. The free energy surfaces (Fig ) reveal the average free energy difference between the minima of gauche-and trans conformations (present in the 1wfc and 3fly structures, respectively) of the apo state to be ∼ 8 ± 2 kJ/mol. This matches well the observed shift in the binding free energies calculated using 1wfc and 3fly starting structures for the apo state. Due to insufficient end-state sampling and high energy barriers, we do not observe a transition in this rotamer during short 10 ns equilibrium simulations, yet simulations started from the true apo state allowed taking the missing ∆G contribution into account.