The overall accuracy of the free energy calculations shows dependence on the quality of the starting ligand poses. For example, the TNKS2 system exemplifies that for the cases where different docking algorithms converge to a consistent result, the subsequent alchemical calculations yield a high accuracy prediction. Such a well behaved set of ligand poses allows zooming in on particular details that affect the free energy estimates. One interesting case is shown in Fig 7 . Here, a clear trend is visible for three poses yielding a better agreement with experiment (colored in blue) than for the other three structures (colored in pink). Whilst several structural differences can be discerned among the structures, notably hydroxyl group direction and pyridine ring orientation, only the pyridine ring placement clearly correlates with the accuracy of the estimated affinities. Thus, this example demonstrates that alchemical free energy calculations can be very sensitive even to the positioning of single atoms. An even more surprising effect is that the presumably incorrect pyridine orientations are predicted to have stronger binding affinities.