The missing amide resonances in the 1H-15N HSQC spectrum of SARS-CoV-2 Nsp9 are likely due to intermediate backbone dynamics on the ms to μs timescale. To probe backbone dynamics on the faster ns–ps timescale and obtain an idea on the “compactness” of the structure, steady-state {1H}-15N heteronuclear NOE ratios were collected in triplicate for Nsp9 at a concentration of 0.5 mM and the mean values plotted in Fig. 5a. Aside from a few residues near the termini and a couple of loops between β-strands, most of ratios are above 0.8, an average suggestive of a relatively rigid overall structure with motion on the fast timescale (ps) restricted. This is in agreement with the compact single domain for the monomer observed in the crystal structure of SARS-CoV-2 Nsp9 composed of one α-helix and seven β-strands arranged into a cone shaped β-barrel. This conclusion based on the heteronuclear NOEs is further corroborated in the backbone 15N T1 and T2 relaxation times plotted for SARS-CoV-2 Nsp9 in Fig. 5b and c, respectively. Most of the T1 values fall between 600 and 800 ms except for a few residues near the termini and most of the T2 values fall between 50 and 100 ms with the exception of a few residues near termini or in loops between β-strands.

Plots of the backbone a {1H}-15N heteronuclear NOEs (cyan) b T1(red), and c T2 (purple) values for SARS-CoV-2 Nsp9 (~ 0.5 mM) collected at a 1H resonance frequency of 600 MHz, 303 K. On top of the plots is a schematic representation of the elements of secondary structure observed in the crystal of SARS-CoV-2 Nsp9 (PDB ID 6WXD): β-strands = magenta, α-helix = orange. Residues with missing or unassigned resonances in the 1H-15N HSQC spectrum of SARS-CoV-2 Nsp9 are identified with red blocks below scheme and residues with weak amide cross peak intensities relative to the majority of amide resonances are identified with cyan blocks

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