It is important to note, however, that the increased sampling does not automatically translate into a better agreement of the simulated trajectory with the experimentally measured observables. For example, longer simulations of the tyk2 kinase in its apo state (4gih 30 with the ligand removed; Fig ) explore a broader range of conformations. However, as simulations progress, they deviate substantially from the starting crystallographic structure. The substantial drift of simulated trajectories, in turn, results in large uncertainties of the calculated binding affinites and deteriorates the ∆G prediction accuracy. This observation indicates that either the longer sampling reaching Figure : The effect of T106 rotamer states of the p38α kinase on the calculated ligand binding ∆G. Free energy surfaces (A) of the χ1 dihedral angle for the T106 residue obtained from well tempered metadynamics simulations starting from 3fly and 1wfc structures as well as from the output of 1 µs equilibrations started from 3fly. χ1 dihedral angle for the T106 residue in the crystallographic apo (1wfc) and holo (3fly) states, as well as in 5 independent simulations of 1 µs each (B). Binding ∆G calculated by initializing apo state simulations with the 1wfc structure (C) and the end-states from 1 µs simulations (D). Dark and light shaded areas represent regions deviating from experiment by at most 1 and 2 kcal/mol. 1µs for each of the 5 repeats is still not sufficient, or the new free energy minima identified by the force field are not representative of the true free energy landscape.