The absolute ∆G calculations are particularly sensitive to the decoupled ligand restraining method. In our setup we employ a rigorous restraining approach 7 acting between the protein and the decoupled ligand to anchor it in a narrow range of orientations within the binding pocket (SI Section 1). This restraint scheme Figure : Diagram of the thermodynamic cycle for absolute binding calculations. As inserting a whole protein around a ligand would involve a massive disturbance in the system, the binding free energy ∆G bind is calculated by traveling the long way along the thermodynamic cycle: first decoupling the ligand from the surrounding solvent, applying the analytical correction for the effect of protein-ligand restraints , and then coupling the ligand back in the protein active cite. The equilibrium structures for the decoupled ligand in the active site (state B) can be generated by aligning its structures in solvent (state B ) into equilibrium frames of the apo protein.