It appears that the calculation of the overall offset is one of the major challenges in the absolute binding free energy estimation. Interestingly, given the equivalent simulation conditions for a set of ligands, even such large overall shifts in the calculated absolute ∆G values may have no effect on the relative free energies between the ligands (e.g. the case of tyk2 in Fig 1 and Fig S2). This suggests that the cause of the offset could be largely the same for all the considered ligands and cancels out in calculating the free energy differences. The protein-ligand complexes investigated in this study present a Figure : Identification of the most likely apo state for pde2 system based on binding affinities of 21 inhibitors. The absolute binding free energies were calculated using 6ezf holo state and 4 structures without the ligand to represent the apo state (4htz 55 , 6ezf , 4d08 and 4d09 8 ). The common holo state allows comparing the apo states one to another in terms of ∆G (the uncertainty of each estimate is below 1 kJ/mol). 6ezf structure is identified as the most likely apo structure based on the binding free energies for the considered ligand set. The panels on the right compare the experimental binding affinities to those calculated with each of the apo structures. Dark and light shaded areas represent regions deviating from experiment by at most 1 and 2 kcal/mol. convenient set of systems for testing this hypothesis: the relative free energies for these systems have been previously calculated directly by alchemical transformations between ligand pairs with a non-equilibrium approach and the same force field .