Protein expression and purification

The proteins used for structural and biochemical studies were overexpressed in E. coli BL21-Gold (DE3) as previously described (Wan et al., 2020). The selenomethionine (SeMet)-substituted protein was produced in E. coli BL21-Gold (DE3) using the metabolic inhibition method as previously described (Doublie, 1997). Cells were collected by centrifugation and kept at –80°C for storage.

Affinity purification was performed using a HisTrap HP column or loose NiNTA-agarose resin (GE Healthcare Life Sciences) at room temperature under strict anaerobic conditions as previously described with minor modification (Wan et al., 2020). For the purification of the WhiB7: σ^A_C82-β_tip complex, a 10 column-volume wash with 50 mM Tris-HCl pH 8.0, 1M NaCl, 1mM DTT was added in the purification procedures to remove the DNA contamination before elution. Imidazole in the eluted fractions was removed by passing through either a desalting column or a Superdex 200 column (GE Healthcare Life Sciences). The samples after each step of purification were analyzed by SDS-PAGE and by UV-Visible (UV-Vis) spectroscopy. Unless otherwise specified, the final purified proteins in 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1mM DTT were stored in liquid nitrogen until use. Protein concentrations were estimated by the Pierce Bradford Assay Kit (Thermo Fisher Scientific) and by the absorption at 280 nm.