The overall binding free energy obtained with this bubble approach is statistically equivalent to the binding free energy obtained with the standard protocol (SI Table ). The uncer-tainty, however, is slightly higher with the bubble approach, partly because four additional legs in the thermodynamic cycle (Figure ) combine to increase the overall uncertainty. 5.5.4. The enhanced sampling technique HREX was not able to overcome inadequate sampling. Enhancing sampling in the EQ approach using HREX did not lead to major improvements with respect to convergence and standard deviation. As shown in Figure , the standard deviation is actually higher using HREX (0.5 kcal/mol) than without enhanced sampling (0.3 kcal/mol), as we explore below.