4.1. We constructed a thermodynamic cycle to calculate the binding free energy. Ligand binding/unbinding events (associationdissociation) are typically too slow for direct simulation. Therefore, it is more convenient to compute binding free energies via a thermodynamic cycle. Free energy is a state function, meaning that this allows us to construct artificial (more convenient) pathways to get between the states of interest. Figure shows the thermodynamic cycle used in this paper. The binding free energy (panel F) is not simulated directly but obtained through summation along the cycle.