Abstract
Heterogeneous and dynamic biomolecular complexes play a central role in many cellular processes but are poorly understood due to experimental challenges in characterizing their structural ensembles. To address these difficulties, we developed a hybrid methodology that combines X-ray crystallography with ensemble selections typically used in NMR studies to determine structural ensembles of heterogeneous biomolecular complexes. The method, termed READ, for residual electron and anomalous density, enables the visualization of heterogeneous conformational ensembles of complexes within crystals. Here we present a detailed protocol for performing the ensemble selections to construct READ ensembles. From a diverse pool of binding poses, a selection scheme is used to determine a subset of conformations that maximizes agreement with the X-ray data. Overall, READ is a general approach for obtaining a high-resolution view of dynamic protein-protein complexes.