Instead of using direct MD simulations to obtain populations, an alternate approach would have been to employ AFE calculations for each distinct binding mode which, in the best case scenario, could be roughly comparable in cost to MD/NCMC implemented via BLUES. Specifically, standard AFE calculations require separate simulations for 12 lambda values with 5 ns of simulation for each lambda value, resulting in a total 60 ns worth of simulation (based on simulation protocol reported by Kaus et al. ).