Fig. 4.

The Protein Prospector parameter “score difference” is the best single classifier of cross-link spectral matches. Cross-linking of RNA pol II by DSS resulted in 2535 spectral matches to the target database, which also corresponded to measurable Cα distances on the crystal structure (blue). 5319 hits to the decoy database (green) were assigned random distance measurements sampled from the set of all possible Lys-Lys distances on the PDB:1WCM structure. The vertical line indicates 25 Å, the nominal span of DSS. The horizontal line indicates a score threshold that classifies 92.5% of decoy matches correctly. A, classification based on XLScore misclassifies many seemingly correct cross-link identifications (those with distance < 25 Å). B, classification based on score difference results in better discrimination between target and decoy matches and leaves fewer apparently true positives misclassified.