Fig. 3: Substrate containing highly favored unpaired U enables reconstruction of Nsp15-bound dsRNA with defined sequence.

A Design of dsRNA oligo incubated with Nsp15 for cryo-EM experiments; identical to unpaired U substrate used in Fig. 2. Nucleotides that were found to be sites of interaction with Nsp15 are labeled with their position number. Labels in the target strand containing the unpaired U are positive, with position 1 at the 5′ end; labels in the complement strand are negative, with position -1 at the complement strand’s 5′ end. B Cryo-EM density map of dsRNA-bound Nsp15 (H235A mut) at a resolution of 3.24 Å, including dsRNA (pink), protomers of Nsp15 that interact with dsRNA (P1, P2, P4 in shades of dark teal), and remaining protomers of Nsp15 (P3, P5, P6 in shades of light blue-gray). C Closeup view of atomic model and mesh cryo-EM density map showing dsRNA residues surrounding the flipped-out U14, which is engaged in the active site of Nsp15 protomer P1. H-bonds are shown in cyan. D Cryo-EM density map of Nsp15-bound dsRNA, colored by local resolution. Protomer P1 is outlined in black. E Residues of Nsp15 protomer P1 calculated (via dr_sasa⁴⁷) to interact with dsRNA, with P1 endoU domain represented as a transparent cartoon and dsRNA density map colored by local resolution. Residues previously shown to be key in Nsp15 cleavage of dsRNA are labeled; see Supplementary Information for an alternate version with all interacting endoU residues labeled. H235 (light gray) has been superimposed from a structure of apo WT nsp15 (PDB ID 7N33) over A235. F Binding pocket of Nsp15 for dsRNA, colored by electrostatic potential (left) and hydrophobicity (right, represented by the molecular lipophilicity potential) using default settings in ChimeraX. Residues calculated to interact with dsRNA are outlined in black and shown in full color according to key; all other residues of Nsp15 are included to provide structural context but shown at 65% transparency. See Supplementary Fig. 5 for alternate version with all residues of Nsp15 in full color.