The standard deviation of free energy estimates as a function of the number of replica exchange cycles, for (a) changing the ligand temperature from 300 K to 600 K, and (b) scaling the receptor-ligand interaction grid from 0 to 1 while decreasing the temperature. Values were calculated between dipropyl disulfide, thianaphthene, isobutylbenzene, dibutyl-disulfide, phenylacetylene, cyclohexane, 1-heptanol, 1-propanol, 1,1-diethylurea, p-xylene, and a snapshot of T4 lysozyme from an alchemical pathway calculation with n-hexylbenzene. The thick black line is the average of the standard deviation across the 10 complexes.