Site Confidence Scores among All Available Structures to Help Infer Conformational Origins of Identified Cross-Links

Cross-linked peptides are indicative of protein conformations in samples due to their reactivity with multiple proximate residues. Since their cross-linked proteins often have alternative conformations, each with available structures, large-scale cross-link data sets require advanced capabilities to readily and accurately assess the likelihood of a cross-link originating from all available structures. Traditionally, the Euclidean or solvent-accessible surface distance (SASD) separating the residues has been used to assess the possibility that a cross-linked product arises from one of many structures. However, solvent accessibility and the potential for salt bridge formation of each reactive residue can also affect the likelihood of cross-linker attachment. Here, we describe the addition to the public cross-linking database XLinkDB of a confidence score that combines SASD, solvent accessibility, and salt bridge information to better assess the likelihood of a cross-link originating from residue pair sites among possible structures. We show how this score correctly predicts the state specificity of cross-links of ADP/ATP translocase, identifying cross-links that can serve as biomarkers of particular protein conformational configurations. These efforts also reveal that 12% of cross-links on XLinkDB have no PDB structures available that yield high confidence scores and thus may have value for additional modeling to predict alternate structures.