Purification of BbZIP Protein Variants

The expression of BbZIP was reported previously^{46 (link)}. In brief, the wild type or the variants of BbZIP was expressed in the strain of C41 (DE3) pLysS (Lucigen) in LBE-5052 Autoinduction medium for 24 h at room temperature. After harvest, spheroplasts were prepared and lysed in the buffer containing 20 mM Hepes (pH 7.3), 300 mM NaCl, 0.25 mM CdCl₂, and cOmplete protease inhibitors (Sigma-Aldrich)^{83 (link)}. n-Dodecyl-β-D-maltoside (DDM, Anatrace) was added to solubilize the membrane fraction at the final concentration of 1.5% (w/v). The His-tagged protein was purified using HisPur Cobalt Resin (Thermo Fisher Scientific) in 20 mM Hepes (pH 7.3), 300 mM NaCl, 5% glycerol, 0.25 mM CdCl₂, and 0.1% DDM. The sample was then concentrated and loaded onto a Superdex Increase 200 column (GE Healthcare) equilibrated with the gel filtration buffer containing 10 mM Hepes, pH 7.3, 300 mM NaCl, 5% glycerol, 0.25 mM CdCl₂, and 0.05% DDM. For the variants with introduced cysteine residue(s), 1 mM TCEP was added during purification but excluded in the gel filtration buffer. The peak fractions were used for crystallization, cryo-EM, or crosslinking experiments.