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.
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Acknowledgment
This work was supported by the National Institutes of Health (R01-GM102829 to J.C.A.B. and K99/R00-GM120388 to S.H.). J.C.A.B. is a Howard Hughes Medical Investigator. The authors would like to thank S. Rocchio for comments on the manuscript and M. Mourao for useful discussions. The authors would also like to thank C. Stockbridge and the LSA-IT development team for the assistance in coding.
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Salmon, L., Ahlstrom, L.S., Bardwell, J.C.A., Horowitz, S. (2018). Selecting Conformational Ensembles Using Residual Electron and Anomalous Density (READ). In: Marsh, J. (eds) Protein Complex Assembly. Methods in Molecular Biology, vol 1764. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7759-8_31
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DOI: https://doi.org/10.1007/978-1-4939-7759-8_31
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