Sample preparation

The nsp3a plasmid was transformed into BL21 (DE3) E. coli cells and the protein expressed heterologously with an N-terminal His₆ tag. Cells were grown at 37 °C until OD₆₀₀ of 0.6–0.8, at which point protein expression was induced with IPTG and incubated for 5 h at 37 °C. Bacteria were harvested by centrifugation and the cell pellet resuspended in buffer A (50 mM Tris–HCl pH 8.0 and 250 mM NaCl) with protease inhibitors (complete, Roche). Cell lysis was performed by sonication, followed by centrifugation (45 min, 18,000 rpm at 5 °C).

The protein was purified by affinity chromatography on Ni–NTA agarose (ThermoFisher), washed with buffer A supplemented with 20 mM imidazole and eluted with buffer A supplemented with 500 mM imidazole. TEV cleavage was achieved by incubation with TEV protease at 4 °C coupled with dialysis into buffer A supplemented with 2 mM DTT, and the protein concentrated and subjected to size exclusion chromatography on a HiLoad 16/600 Superdex 75 column (GE Healthcare) in NMR buffer (50 mM Na-phosphate, pH 6.5, 150 mM NaCl). For ¹⁵N and ¹³C isotope labelling, cells were grown in M9-minimal medium supplemented with ¹⁵N-NH₄-Cl and ¹³C₆-d-glucose (1 g/L each).