Abstract
Chemical cross-linking coupled with mass spectroscopy (CXMS) is a powerful technique for investigating protein structures. CXMS has been mostly used to characterize the predominant structure for a protein, whereas cross-links incompatible with a unique structure of a protein or a protein complex are often discarded. We have recently shown that the so-called over-length cross-links actually contain protein dynamics information. We have thus established a method called DynaXL, which allow us to extract the information from the over-length cross-links and to visualize protein ensemble structures. In this protocol, we present the detailed procedure for using DynaXL, which comprises five steps. They are identification of highly confident cross-links, delineation of protein domains/subunits, ensemble rigid-body refinement, and final validation/assessment. The DynaXL method is generally applicable for analyzing the ensemble structures of multi-domain proteins and protein–protein complexes, and is freely available at www.tanglab.org/resources.
Highlights
Chemical cross-linking coupled with mass spectroscopy (CXMS) has been used to characterize protein structures
Cross-linking reagents can react with specific amino acids in a protein, and two amino acids separated by a distance shorter than the 100 | December 2017 | Volume 3 | Issues 4–6
Mass spectrometry analysis is used to identify the cross-linked residues, which can be translated to inter-residue distance (Kahraman et al 2013; Lossl et al 2014)
Summary
Chemical cross-linking coupled with mass spectroscopy (CXMS) has been used to characterize protein structures. CXMS analysis of protein ensemble structures length of the cross-linker can be theoretically crosslinked (Fig. 1). Xplor-NIH (Schwieters et al 2003), the software package for biomolecular structure refinement against experimental and knowledge-based restraints, is used here to identify an optimal ensemble structure that can account for all CXMS restraints. (3) To define domain boundaries; (4) To validate domain definition and evaluate local flexibility; (5) To classify and identify the cross-links (intradomain vs inter-domain, intramolecular vs intermolecular cross-links), and prepare the CXMS restraints table; (6) To prepare the starting structure files for XplorNIH; (7) To patch the cross-linker to the protein structure; (8) Ensemble rigid-body refinement against the CXMS restraints; (9) Cross-validation with a subset of cross-links; (10) To analyze and validate with other types of data
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