We investigated different sampling strategies for protein model refinement within a previously established framework (see Figure ). Starting from an initial model, a common pre-sampling stage consists of the application of locPREFMD to resolve stereochemical errors such as severe atomic clashes, cis-peptide bonds, and poor rotamer states that may cause problems during simulations. The initial model after locPREFMD was then solvated in an explicit water box, and equilibrated by heating up to a chosen simulation temperature. The subsequent sampling stage involved MD simulations according to different strategies as described below with the goal of generating an ensemble of structures. During the postsampling stage, again common to all protocols, a set of structures was selected among the sampled structures to be ensemble averaged. We used two ensemble selection schemes: (1) the 25% lowest-scoring conformations according to RWplus 39 were selected; (2) a combined score based on RWplus and the deviation from the initial structure was applied to select the sub-ensemble. The selected structures were then averaged based on Cartesian coordinates. Stereochemical problems due to averaging were removed via local relaxation using short MD simulation, followed by SCWRL4 to rebuild side chains, and again the application of locPREFMD to polish the quality of the final models.