Another difference in the TI and FEP results may stem from the perturbative nature of FEP. If the phase space overlap of alchemical states is small, then the FEP result may be unreliable. A quantitative measure of this overlap of states can be made with an overlap matrix which was computed for all transformations and thermodynamic legs. The overlap matrix is described in detail elsewhere but, briefly it is a matrix of rank K × K, where K was previously defined in that work as the number of alchemical states. Each entry in the matrix is the probability that a sample from a given alchemical window λ i could have been sampled from some other alchemical window λ k . For reliable free energy calculations it has been proposed in previous work that the overlap matrices should be tri-diagonal with offdiagonal values greater than 0.03 . When the overlap matrices are averaged across replicas all but one of the FEP calculations performed in this work satisfied these conditions and this result is shown in figure . The simulation with the abnormal matrix is the complex leg of an OpenMM simulation for the MCL1 target. The abnormal transformation is named l12-l35;