E coli bl21 codonplus de3 rp competent cells

His-tagged RBD (217-516) was expressed in E.coli BL21-CodonPlus (DE3)-RP competent cells (Agilent) and purified by nickel exchange chromatography. RBD was further purified by size exclusion chromatography (Superdex 200 column). The TBE RNA construct was prepared by annealing two individual strands, each containing one side of Stem II (Fig. 1b). (Strand A: 5’ UUCAUUCAGUUCU 3’ Strand B: 5’ UAGAACUGUCAUU 3’). To anneal the construct, the two strands were heated to 95°C for 3 minutes in annealing buffer (500 mM NaCl, 20mM Tris-HCl pH 7.8) and then allowed to slowly cool to room temperature. After cooling to room temperature MgCl₂ and DTT were each added to a final concentration of 1mM. The tTRBD protein was mixed with the annealed TER-TBE RNA at a stoichiometry of 1:1.5 in high salt buffer (500mM NaCl, 20mM Tris-HCl pH 7.8, 1mM MgCl₂ and 1mM DTT). This mixture was then dialyzed overnight (~16 hours) into low salt buffer (100mM NaCl, 20mM Tris-HCl pH 7.8, 1mM MgCl2 and 1mM DTT). Formation of protein-RNA complex was analyzed using diagnostic sizing chromatography on a Superdex 200 column (Supplemental Fig. 3c) prior to setting crystallization trays.