Reliable candidate discovery is a major bottleneck in the drug development pipeline. Identification and validation of compounds in preclinical trials span many years with thousands of potential candidates whittled down to a single approved drug. Modern developments to in silico methods seek to remedy the time and cost drain of early stage discovery, providing information on ligand properties without the need for time-and resource-intensive experiments. In particular, relative binding free energy (RBFE) calculations have been widely adopted due to their ability to provide thermodynamic information on potential drug candidates while only requiring simulation data generated from molecular dynamics (MD). The lysozyme of the T4 bacteriophage serves as an ideal test system for binding free energy calculations. A unique leucine to alanine mutation (L99A) creates a solvent-inaccessible and virtually apolar binding pocket with high binding rates for small, organic molecules. The properties of the binding pocket prevent motion of bulk water in and out of the binding pocket, decreasing computational cost and increasing the ease of performing RBFE calculations. However, when bound to a congeneric series of alkyl benzenes, the T4 L99A binding pocket is experimentally observed to adopt three discrete conformations (Figure ), each with increasingly more area open to bulk solvent. The emergence of several discrete binding pocket states upon binding of congeneric ligands has been observed in other systems as well, suggesting that many protein binding pockets may undergo significant changes for even relatively small ligand perturbations. Here we show three overlays of the F-helix region from the crystal structures we use to represent each state in this work. Shown is the benzene-bound crystal structure (4w52) to represent the experimentally defined closed state (purple), the butylbenzene-bound crystal structure (4w57) to represent the experimentally defined intermediate state (cyan), and the hexylbenzene-bound crystal structure (4w59) to represent the experimentally defined open state (green).