The relative binding free energy difference between ligand 1 (L1) and ligand 2 (L2) can be expressed as DDGbinding = DGbinding (L2) -DGbinding (L1) = DGbound (L1 to L2) -DGfree (L1 to L2), where both the bound and the unbound, i.e. free ligand in solution, are considered in a thermodynamic cycle. The change in the area under the curve of dV/dl is obtained via Gaussian quadrature. Here, a 5-point Gaussian quadrature is calculated, using the five l-values. For each transformation, 10 ns were run at each l-value for the complex and the free system. The cumulative average for each was then plotted against time to determine the convergence: The quality of the thermodynamic estimates and the correlation with experimental data improves when only considering the cumulative average of the last 4 ns (data not shown).