Cross-Linking-Mass Spectroscopy.

Purified RDR2_771–970 and NRPD1_1–300 proteins were preincubated then cross-linked using 0.1 mM Bissulfosuccinimidyl suberate (BS3). Gel-purified cross-linked protein complexes were reduced using 10 mM TCEP (Tris(2-carboxyethyl)phosphine), alkylated with 20 mM iodoacetamide, and digested for 16 h with Trypsin or Chymotrypsin, at two concentrations (0.1 mM and 0.5 mM) for each enzyme. Resulting peptides were resolved by HPLC using a C18 column and an acetonitrile gradient and subjected to electrospray ionization and analyzed using an Orbitrap Fusion Lumos mass spectrometer. Details are provided in SI Appendix. Resulting data have been deposited at the ProteomeXchange Consortium via the PRIDE partner repository (38) with the dataset identifier PXD020170.