Fig. 2. 5′-UMP coordination by the Nsp15 endoU active site.

a Amino acid sequence alignment of endoU active site residues from Nsp15 homologs. Secondary structure motifs observed in the nucleotide-bound Nsp15 cryo-EM structure are shown above with their corresponding amino acid residue boundaries. b Nucleotide-bound Nsp15 cryo-EM map reconstruction with protomers colored as seen in Fig. 1b. Excess UTP was added to the sample resulting in additional density within all six endoU active sites. The nucleotide density is colored in cyan and the black box demarcates the endoU active site. c Nsp15 coordination of 5′-UMP ligand. Due to the poor density of the UTP β- and γ-phosphates, 5′-UMP was modeled into the active site. Cartoon model of the 5′-UMP-bound Nsp15 endoU active site where 5′-UMP (cyan) and individual residues H235, H250, K290, S294, and Y343 are shown as sticks (top). Model of uracil base discrimination shown as sticks (bottom). d RNA cleavage activity of Nsp15 variants (2.5 nM) incubated with FRET RNA substrate (0.8 μM) over time. RNA cleavage was quantified from three technical replicates. The mean and standard deviation are plotted and P values of wt-Nsp15 compared to H235A (blue; P < 6.4 × 10⁻⁹) and H250A (red; P < 6.2 × 10⁻⁹) are reported from two-tailed Student’s t tests.