We compared the performance of the EQ and NEQ methods on two different protein targets, examining two ligands binding to the T4 lysozyme mutant L99A and a ligand binding to HSP90. Each system has its own challenges which we will introduce in this section. 3.1. The T4 lysozyme mutant L99A is a simple test system, but still has sampling challenges. The T4 lysozyme mutant L99A is a simple model binding site that has been extensively studied both experimentally and computationally. The leucine to alanine mutation leads to the formation of an apolar cavity that binds small molecules. We chose this system for several reasons. First, the dry binding site and the simple, mostly rigid ligands make the system appropriate for the comparison of methods, as sampling times are not excessively long. Secondly, since it is a well studied binding site, it is possible to compare the results with other methods. Lastly, it poses some interesting challenges for alchemical FEC. Toluene is known to bind with two different binding modes (Figure ). If the population or free energy of each pose is assessed separately, the crystallographic pose should be the more favorable. Other ligands, such as 3-iodotoluene and pxylene, induce slow side chain rearrangements upon binding. In particular, the valine 111 side chain rotates to make room for the slightly larger ligands (Figure ). Here, it will be interesting to see if this slow motion can be cap-tured in the fast non-equilibrium transitions and whether correct binding free energies can be obtained. 3.2. HSP90 is a challenging, pharmaceutically relevant target. Even though simple systems, for which convergence can be achieved on reasonable timescales, are highly valuable for method comparison, we are ultimately interested in how the methods perform on pharmaceutically relevant systems. HSP90 is an anticancer drug target, and multiple inhibitors of the protein have been developed. Computationally, it is a more challenging system than the T4 lysozyme L99A mutant, though still regarded as relatively straightforward on the spectrum of potential pharmaceutical targets. The binding site is solvent exposed and, relative to T4 lysozyme L99A, the ligands are larger, more polar and flexible (Figure ). While some ligands introduce larger protein conformational changes upon binding, 50 these are not observed for the ligand considered in this study.