(a) The capping mechanism of viral mRNA in SARS-CoV-2 involves four proteins: NSP13 (helicase, orange), NSP14 (green) and NSP16 (pink) and NSP10 (blue). NSP10 acts as a cofactor for both NSP14 and NSP16. (b) The structure of NSP10 (PDB entry 6CZT). NSP10 (white/gray cartoon) in complex with (c) NSP14 (cyan) and (d) NSP16 (green). (e) Overlay of NSP10 interacting with NSP14 and NSP16 displaying the partially overlapping interface of both enzymes with NSP10.

Summary of the top three variants occurring in NSP10 calculated using Dynamut2. The percent frequency and effect on calculated protein stability are shown. A summary of the top 39 mutations have been listed in Supplementary Table S3. Only mutations with a count larger than 1000 and a percent frequency larger than 0.01% have been included.

Data collection, data processing, and model refinement statistics for SARS-CoV-2 NSP10 variant T102I. Data in parenthesis correspond to the highest resolution shell.

(a) Mutation count of the top 39 mutations in NSP10 extracted from >13 million WGS. (b) Locations of top six mutations on the NSP10 structure. Mutation positions labeled in red are the most frequently occurring and were used in crystallographic studies. (c) The spatial position of the mutations relative to NSP14 and NSP16 structures. Local structural environment of (d) T12I, (e) T102I and (f) A104V mutations (blue sticks) superimposed on the wild-type structure (white). The red discs represent regions where pairwise overlap of van der Waal radii occurs between the side chain atoms of the point mutation and the surrounding structural elements. The green discs represent regions where atoms are almost in contact.

Biochemical characterization of native NSP10 and the three most frequent variants. (a) Melting curve of native NSP10 and its three variants. The red, yellow, green and blue curves show the data measured for native NSP10, T12I, T102I and A104V variants, respectively. (b) Derivative curves of NSP10 and its three variants using the same color code. (c) MST dose response curves for native NSP10 and its variants in the presence of NSP14 ExoN domain. The green, red, blue and brown curves represent native NSP10, T12I, T102I and A104V, respectively. (d) MST dose response curves for native NSP10 and its variants in the presence of NSP16, with the same color codes as above. All experiments were conducted at least in triplicate.

Structural details of the NSP10 T102I variant. (a) 2Fo-Fc map (1σ) of the NSP10-T102I variant. (b) Magnification of the I102 side chain in the electron density map. (c) Structural overlay of wild-type NSP10 (PDB entry 6ZCT; blue) and variant T102I (green). The position of T102I is illustrated as sticks (d) Overlay of T102 (blue) and I102 (green) side chains. The position of a water molecule present in wild-type NSP10 that makes interactions with the T102 side chain is shown. Zinc atoms are illustrated as grey spheres and water molecules are illustrated as red circles.

Well-tempered metadynamics reveal similar conformational dynamics for the wild-type and variant NSP10. Free energy surface plots of the (A) wild-type T12 and T12I variant, (B) wild-type T102 and T102I variant and (C) wild-type and A104V variant. The wild-type and the variants explore similar conformational landscapes. An overlay of the structures extracted from the largest populated minima highlights similar conformations. The crystal structure 6ZCTXTAL (yellow), the representative conformation extracted from the largest minima in the wild-type simulation (slate blue) and the mutant simulation (green) are superimposed. The resolved crystal structure conformations are extrapolated on the minima and illustrated as a star (yellow-6ZCTNSP10; green - 7MC5NSP14; pink - 6W4HNSP16; cyan - T102IXTAL)

Mutational propensities of various SARS-CoV-2 genes. The number of mutations observed in each gene at the protein sequence level and normalized by sequence length is depicted (as blue bars). The genes are ranked in order of their mutational propensity.