Fig. 4: Structure probing by CLMS.

a Map of hetero-protein crosslinks observed in RNAP^ΔδΔHelD-δ-HelD complex. b Crosslinks identified in RNAP^ΔδΔHelD, RNAP^ΔδΔHelD-δ, RNAP^ΔδΔHelD-HelD, and RNAP^ΔδΔHelD-δ-HelD. Binding of both δ and HelD leads to strongly reduced crosslinking between β and β′. c Distribution of Cα-Cα distances between crosslinked residue pairs in reference to the RNAP-δ-HelD structure. Crosslinks with Cα-Cα distances within 25 Å, the theoretical crosslinking limit of sulfo-SDA, green; crosslinks with Cα-Cα distances >25 Å, magenta; distance distribution of random residue pairs in the RNAP-δ-HelD structure, gray. d Numbers of crosslinks (bars) between β and β′ identified from the four cross-linked complexes, and fractions of over-length crosslinks (percentages at the bottom). Crosslinks are color-coded as in b. In the RNAP^ΔδΔHelD-δ-HelD complex, a significantly reduced number of β-β′ over-length crosslinks (in reference to the RNAP-δ-HelD structure) compared to the RNAP^ΔδΔHelD, RNAP^ΔδΔHelD-δ, and RNAP^ΔδΔHelD-HelD complexes suggests that δ and HelD cooperate to stabilize an open conformation of RNAP. e Comparison of β-β′ crosslinks observed with RNAP^ΔδΔHelD, RNAP^ΔδΔHelD-δ, RNAP^ΔδΔHelD-HelD, and RNAP^ΔδΔHelD-δ-HelD. The green boxed region, crosslinks between the β1/2 lobes (residues 146–248) and the β′ shelf and jaw (residues 794–1141) observed in the first three complexes but almost absent in RNAP^ΔδΔHelD-δ-HelD. f Structure of the RNAP-δ-HelD complex highlighting the β1/2 lobes (lemon green) and β′ shelf and jaw (forest green), which largely lack crosslinks in the presence of δ and HelD (green box in e).