Among the protein-ligand complexes investigated in this work, 6 out of 7 systems have both their apo and holo structures resolved by means of x-ray crystallography. We have probed two methods of calculating the binding ∆G value: firstly, removing the ligand from the holo state and treating the obtained structure as an apo state. For the second approach we used the crystallographically resolved apo structure directly. Overall, there is a large and significant improvement in the calculated binding ∆G accuracy when an experimentally defined apo state is considered explicitly (Fig 2 ). A substantial improvement in the AUE (from 7.1±0.6 to 4.4±0.5 kJ/mol) shows that starting the simulations with a corresponding apo structure largely removes an offset which is otherwise present for the calculations initialized with the holo structures only. This indicates that substantial rearrangements occur in the studied proteins upon ligand binding that do not equilibrate at the nanosecond timescale covered in the simulations.