Using the same amount of sampling, the two approaches converged within uncertainty to the same values for toluene and 3-iodotoluene. The methods agreed to within 0.2 kcal/mol of each other fairly quickly (∼40 ns total simulation time). In the case of the HSP90 compound, it took ∼200 ns to agree within 0.2 kcal/mol, and EQ and NEQ free energy estimates did not converge to statistically the same result, but were very close (∆∆G=0.14 kcal/mol). When running longer (500 ps) but fewer NEQ switching transitions the methods agreed, but the standard deviation was slightly higher (SI Figure ). For these three ligands, the NEQ approach was able to reduce the standard deviation and the bias faster than the EQ approach, suggesting that it is more efficient. However, this needs investigation on a larger set of molecules. 5.2. Toluene binding to T4 lysozyme mutant L99A. With the force field used here, toluene binds to the cavity in this T4 lysozyme mutant with two (non-symmetric) binding modes (Figure ). The crystallographic binding pose (pose I) has a more favorable binding free energy than the other pose (pose II) and interconversion between these two binding modes is very slow compared to typical simulation timescales (e.g. 100 ns or more). Therefore we ran separate calculations for each pose and applied restraints to keep the ligand from transitioning between binding modes (SI Figure ).