Ensemble optimization method (EOM). Rg parameter distributions for wild type (WT, upper panel) and a disulphide-stabilized mutant (MUT, lower panel) of urokinase plasminogen activator protein (SASBDB IDs SASDAT4 and SASDAU4, respectively; Mertens et al., 2012). The distributions of a pool of 10 000 randomized conformations, preserving individual domain structure, are shown as broken lines. The distributions of optimized ensembles selected by the genetic algorithm are shown as blue (WT) and red (MUT) bars, respectively. The decreased width of the distribution of selected structures for mutant relative to wild type indicates a reduction in flexibility, and the observed shift to smaller Rg values provides evidence of structural compaction. The metrics Rflex and Rσ are calculated from the distributions as 82% and 1.0 (wild type), and 45% and 0.1 (mutant). The Rflex value of the random pool is calculated as 85%.