*4.4. Energy Minimization*

The best 20 structures from the PONDEROSA C/S modeling were energy minimized first to remove the few (7.5 on average per each structure) bad contacts present, for 2000 minimization steps, using the steepest descent minimization algorithm. Since the solvent was not present at this stage, the GBSA implicit solvent model was adopted as implemented in the NAMD simulation software [51] according to the model by Onufriev, Bashford and Case [52]. Energy minimization resulted in structures devoid of bad contacts (according to the software Procheck [53]), except for two structures for which bad contacts persisted even after lengthening the minimization to 10,000 steps. The latter two structures were removed from the ensemble for MD simulations. At the same time, the ensemble of the ten most similar structures after energy minimization was retained as representing the NOE-restrained best cluster.
