We next applied the BAT.py binding free energy script to each of the five poses from the above MD equilibration step, using the simultaneous decoupling and recoupling approach . This calculation involves computing the following free energy components: attachment of receptor conformational restraints with ligand in binding site; attachment of conformational restraints to the bound ligand; attachment of translational and rotational restraints to the bound ligand; simultaneous decoupling and recoupling of ligand charge interactions; simultaneous decoupling and recoupling of ligand LJ interactions; release of ligand translational and rotational restraints for the ligand in bulk solvent, leaving ligand freely rotating and effectively at standard concentration; release of ligand conformational restraints in bulk solvent; and release of receptor conformational restraints for the receptor without the bound ligand. For each component, a series of 10 to 16 independent parallel simulation windows, covering the range of restraint weights or transformation lambda values, were run and were analyzed by MBAR or thermodynamic integration, except that the free energy of releasing the ligand translational and rotational restraints was evaluated analytically . Summing the free energy contributions from each component gives the ABFE of the ligand in the simulated pose. The same simulation procedures were applied independently to each pose generated by the MD equilibration stage described above, giving five binding free energy results for each compound, and the overall binding free energy of the compound was obtained by combining the result for all five poses according to Equation 1 .