Purification of His-tagged Acl4 and RpL4

His₆-SUMO-tagged Acl4 and His₆-SUMO-tagged RpL4, encompassing residues 28–361 and 1–277, respectively, were coexpressed, as previously described10 (link). Filtered lysate was applied to a Ni-NTA column (Qiagen) equilibrated with a buffer containing 20 mM TRIS (pH 8.0), 500 mM NaCl and 5 mM β-ME and eluted with a linear imidazole gradient. Protein-containing fractions were pooled and cleaved with ULP1 and dialysed against a buffer containing 20 mM TRIS (pH 8.0), 100 mM NaCl and 5 mM β-ME. Dialysed proteins were applied to a Ni-NTA column equilibrated with a buffer containing 20 mM TRIS (pH 8.0), 100 mM NaCl and 5 mM β-ME and the unbound fraction was loaded onto a HiTrapQ HP (GE Healthcare) ion exchange column equilibrated in a buffer containing 20 mM TRIS (pH 8.0), 100 mM NaCl and 5 mM DTT and eluted with a linear salt gradient. Protein-containing fractions were pooled, concentrated and injected on a 16/60 HiLoad Superdex 200 size exclusion column (GE Healthcare) equilibrated with a buffer containing 20 mM TRIS (pH 8.0), 100 mM NaCl and 5 mM DTT. Protein-containing fractions were pooled, concentrated to ∼20 mg ml⁻¹ and used for crystallization.