where Gi is the free energy of component i which corresponds to either complex (com), protein (pro), or ligand (lig), and is calculated from a set of structures from MD simulations. The free energies can be broken down into a number of components, including the molecular mechanics energy in the gas phase and the solvation free energy. While the former is derived from the molecular modelling forcefield used, the latter is estimated as the sum of the electrostatic solvation free energy calculated using the Poisson-Boltzmann equation and the nonpolar solvation free energy calculated from the solvent accessible surface area. The binding free energy is calculated from the difference between calculations performed for the complex, ligand and receptor conformations obtained from simulation. The 1-trajectory approach was used here, in which conformations of the protein and the ligands were extracted from the ligand-protein complex simulations.