Near-Native Selections from Docking Decoys Figure 1 The binding affinity as a function of rmsd (A˚) for the original ZDOCK decoys (a), after sidechain optimization (b), and after 50 steps of minimization(c). Only top 500 ranked decoys for each case are shown in this figure. This is the result of target 1DFJ. affinities of near-native structures close to that of experi- mental-binding affinity whereas 100 or 200 steps of mini- mization produces the value of binding affinity that is much lower than the corresponding experimental value (20 of 48 targets have known binding affinities).41 Sixteen of the 20 targets have native like complexes in the decoy sets and the mean value of their experimental-binding free energy is 12.6 kcal/mol. This is essentially the same as the calculated value, 12.7 kcal/mol, of the best ranked near-native decoys of the 16 targets after minimized 50 steps. The corresponding values after 100 and 200 steps are 15.8 and 20.3 kcal/mol, respectively). This explains the highest success rate made by 50 steps of minimization because the parameters of the reference state in EMPIRE are trained by experimental-binding affinities. In other words, a stronger interface reference value would be needed to counter the over-reduction of energy for 100 or 200 steps of minimization (for details, see Discussion). It should be emphasized that the parameters in the ref- erence state are trained by the native structures and exper- imental-binding affinities. A 50-step minimization is the best minimization protocol for decoy refinement because it produces binding affinities similar to experimental- binding affinities. However, one certainly can optimize parameters based on minimization protocol as well (see Discussion). To further illustrate the importance of the reference state, we plot the binding affinity as a function of rmsd (Fig. 3) and buried solvent accessible surface area of inter- face (Fig. 4) for target 1ATN. In the absence of reference state, near-native structures (rmsd < 2.5 A˚ ) do not have low-binding free energies. The lowest energy conforma- tions have rmsd values that are greater than 10 A˚ . After Figure 2 The number of successful predictions with or without the reference state as labeled for original ZDOCK decoys, after sidechain optimization, after further 50, 100, and 200 steps of minimization. Figure 3 As in Figure 1, but compares the binding affinity for EMPIRE with or without the reference state (target 1ATN). Only top-ranked 500 decoys are shown. DOI 10.1002/prot PROTEINS 249