Three independent values of ΔΔG were first calculated. Each value of ΔΔG required the calculation of 6-8 alchemical free energies (Fig. , Methods). Repeats of the alchemical free energy components exhibiting the greatest variation were then run to efficiently reduce the confidence limits of the prediction as described in the Methods. First let us consider the overall values of ΔΔG and whether successful predictions can be made.