Finally, titrations of MUP1, which binds a diverse set of fragments, returned a good correlation with experimental data and an almost perfect correlation with FEP results. Interestingly, some of the fragments that bind to MUP1, such as octanol, have many degrees of freedom, and it is reassuring to see these sampled sufficiently. Fig. : Calculated binding free energies from titration calculations vs. experiment and FEP results, using Boresch restraints. Top: T4L99A, Bottom: MUP1. For T4L99A, ABFE calculations were only performed for fragments with a published bound protein crystal structure, whereas for MUP1, the most populated binding modes from the GCNCMC titrations were used. For phenol binding to T4L99A, only a lower limit of the experimental binding free energy is known, as denoted by a large error bar to the right. Crucial to these results, is that as distinct, and symmetrically equivalent, binding modes are sampled naturally by GCNCMC, there is no need to perform more than one set of GCNCMC calculations, or to apply post-hoc symmetry corrections, as is required in ABFE methods. Second, this method of calculating free energies does not require the use of artificial restraints, which can become problematic and often require a large degree of user input. 3 Summary