While the large loop motion has no substantial effect on the ∆G accuracy, a single rotamer flip appears to be responsible for the larger than 9 kJ/mol shift in calculated ∆G. The crystallographic structures 3fly (holo) and 1wfc (apo) Figure : A detailed investigation of the p38α protein-ligand complexes. The apo (1wfc , orange) and holo (3fly, blue) structures have several structural differences close to the ligand binding site: a substantial loop motion and a different T106 rotamer state. In the simulations, the rotamer T106 retains its initial state: shown in lines, with a sphere marking threonine's oxygen. The calculated ∆G values depend strongly on the starting structure (holo or apo) that is used to initialize protein simulations in its apo state: scatterplots at the bottom. The green structure in the sub-panel and corresponding ∆G scatterplot depict a case, where apo simulations were initialized with a holo structure (ligand removed), but with the T106 rotamer set into its apo state. Dark and light shaded areas represent regions deviating from experiment by at most 1 and 2 kcal/mol. have different threonine 106 (T106) rotameric states. Initializing apo simulations with either the experimentally resolved apo structure or a holo structure with the ligand removed yields ensembles where the rotamer never crosses the barrier and remains in its starting state (Fig ). The barrier crossing for the T106 sidechain rotamer appears to be too high to be sampled in the short (10 ns) equilibrium simulations used in the free energy calculation protocol.