The major conceptual difference between the absolute and relative binding free energy calculations stems from the need to explicitly consider the apo protein state when computing absolute ∆G. This poses a challenge for a theoretically rigorous treatment of the decoupled ligand that subsequently needs to be coupled to the system in a well-defined binding site of the protein. In the current work we present a novel approach for the construction of the decoupled ligand state ensembles (see Methods) which, in combination with the ligand restraining protocol , provides an efficient solution to the problem. In brief, our method positions and restrains the decoupled ligand in the binding pocket of the apo protein creating a decoupled state ensemble without the need to explicitly simulate it. Furthermore, explicit consideration of the protein's apo state also requires accurate quantification of a transition between the protein's conformational states sampled upon ligand binding. The non-equilibrium free energy calculation approach presents a convenient setting, where the simulations for holo and apo states can be initialized with different starting structures . In such a way, the apo and holo state ensembles can be generated by simulations started with the corresponding experimentally resolved structures whenever they are available. The initialization of the simulations with a proper starting structure has a profound effect on the accuracy of estimated ∆G (Fig . This observation, however, could be interpreted merely as a sampling issue: routine free energy calculation protocols use short (5-20 ns) equilibrium simulations that may not be sufficient for generating a representative apo state ensemble. Inaccuracies in the estimated free energies due to undersampling have been previously reported for both rel-ative and absolute 5 protein-ligand binding free energy calculations. The issue can be alleviated with longer simulations or enhanced sampling. This appears to be feasible in the case of p38α kinase, where longer simulation of the protein's apo state was able to recover the experimentally resolved rotamer T106 which proved essential for accurate ∆G calculations (Fig ). Yet, the case of tyk2 kinase, for which long (1 µs) simulations were used for the apo state, demonstrates that the extended sampling does not necessarily lead to higher accuracy in ∆G estimation (Fig ). This is in line with several previous observations where enhanced sampling showed no improvement in the accuracy of the free energy estimates. In fact, a deterioration in prediction accuracy can be observed in longer or enhanced-sampling simulations, when the ligand explores poses that are less relevant for binding. In turn, this manifests in an underestimation of the relative binding free energy differences , which we have also observed in our study (Fig 6).