Paramagnetic probe sampling as sensed by the PRE data during different simulated annealing protocols. Only the oxygen atom of the nitroxide group is shown (sphere), against the backbone conformation of bound, closed GlnBP (PDB ID: 1WDN), displayed with the large domain at the bottom. In green are atomic positions optimized assuming the fixed closed conformation. In yellow and red are positions optimized with a flexible hinge, using a 1- or 3-conformer representation of the probe, respectively. All PREs were used (green, yellow, and red). In blue are positions optimized with intra-domain PREs only, assuming a fixed, open conformation. In all cases, results of the 10 lowest PRE energy optimizations are shown (out of 200). Views A and B differ by a 90° rotation.