Lysozyme L99A/M102Q mutant (ABFE), and PLpro (RBFE and ABFE). CDK2 and Lysozyme are common benchmarking systems, allowing us to compare the results from our optimized protocols against published alternatives. The PLpro protein is a more flexible and less well characterized system, thus offering a more difficult challenge for both ABFE and RBFE. For this system, we compare our optimized protocols to long-run protocols ranging from 10-100 ns per λwindow. In total, we evaluated six different implementations of our algorithm: Protocols A-E, and C12, summarized in Table .