Protein Production and Purification

For expression of A. fulgidus or M. mazei Csx3, the standard protocol described in detail previously was followed (Rouillon et al., 2019). Briefly, a culture was grown in 2L Luria-Broth at 37°C to an OD₆₀₀ of 0.8 AU with shaking at 180 rpm. Protein expression was induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside and cells were grown at 25°C overnight before harvesting by centrifugation. For protein purification the cell pellet was resuspended in lysis buffer containing 50 mM Tris-HCl 7.5, 0.5 M NaCl, 10 mM imidazole and 10% glycerol supplemented with EDTA-free protease inhibitor tablets (Roche; 1 tablet per 100 ml buffer) and lysozyme (1 mg/ml). Cells were lysed by sonication and the lysate was ultracentrifuged at 40,000 rpm (70 Ti rotor) at 4°C for 35 min. The lysate was loaded onto a 5 ml HisTrap FF Crude column (GE Healthcare) equilibrated with wash buffer containing 50 mM Tris-HCl pH 7.5, 0.5 M NaCl, 30 mM imidazole and 10% glycerol. Unbound protein was eluted with 20 column volumes (CV) of wash buffer prior to elution of His-tagged protein using a step gradient of imidazole (holding at 10% for 3 CV, and 50% for 3 CV) of elution buffer containing 50 mM Tris-HCl pH 7.5, 0.5 M NaCl, 0.5 M imidazole and 10% glycerol. The His-tag was removed by incubating concentrated protein overnight with Tobacco Etch Virus (TEV) protease (1 mg per 10 mg protein) while dialysing in buffer containing 50 mM Tris-HCl pH 7.5, 0.5 M NaCl, 30 mM imidazole and 10% glycerol at room temperature. Cleaved protein was passed through a 5 ml HisTrapFF column and further purified by size-exclusion chromatography (S200 16/60 column; GE Healthcare) in buffer containing 20 mM Tris-HCl pH 7.5, 0.125 M NaCl. After SDS-PAGE, pure protein was pooled, concentrated and stored at −80°C. H60A, D69A, R71 and H80 variants of A. fulgidus Csx3 (Figure 1—figure supplement 1) were generated using the QuikChange Site-Directed Mutagenesis kit as per manufacturer’s instructions (Agilent Technologies) and purified as for the wild-type protein.