Superdex 75 10 30 column

For crystal formation, non-tagged NbALFA was incubated with a 1.2-fold excess of ALFA peptide (Acetyl-PSRLEEELRRRLTEP-Amide; Genscript) for 60 min on ice. The complex was concentrated to ~25 mg mL⁻¹ and separated from the excess peptide by gel filtration on a Superdex75 10/30 column (GE Healthcare) equilibrated with 20 mM HEPES pH 7.4, 100 mM NaCl, 10% glycerol.
Crystals of the NbALFA-ALFA peptide complex were found in the F2 condition of the JCSG+ screen (Molecular Dimensions). This hit was further optimized using the Hampton Research Additive Screen. A crystal suitable for data collection was found in a drop containing 100 nL of precipitant solution (100 mM sodium citrate pH6.0, 2 M ammonium sulfate, 4% v/v tert-Butanol) and 100 nL of protein solution (10.1 mg mL⁻¹ NbALFA-ALFA peptide complex, 20 mM HEPES pH7.4, 100 mM NaCl, 10% glycerol) in a sitting-drop crystallization plate. X-ray diffraction data collection was performed at BL14.2 at the BESSY II electron storage ring operated by the Helmholtz-Zentrum Berlin, Germany⁴² . The data were processed using DIALS^{43 (link)}, molecular replacement was performed with Phaser^{44 (link)} using the structure with PDB code 5VNV as an atomic model. The structure was built using Phenix Autobuild^{45 (link)} and Coot^{46 (link)}. The structure was refined using Refmac5^{47 (link)}.

WT Erv1 and tryptophan mutants were expressed in E. coli strain Rosetta-gami™ 2 (Novagen) and purified as previously described [17 (link),25 (link)]. Briefly, protein was expressed in the presence of 0.5 mM IPTG and 10 μM FAD at 16 °C for 16–20 h. Induced cell pellets resuspended in buffer A (150 mM NaCl, 50 mM Tris/HCl, pH 7.4) containing 5 mM imidazole, 50 μM FAD and one tablet of EDTA-free protease inhibitor cocktail (Roche) were lysed by sonication on ice. The supernatant fraction containing LE(H)₆-tagged Erv1 was bound to 2–3 ml of Ni²⁺-charged histidine. Bind resin (Novagen) pre-equilibrated with binding buffer. Following a washing step with 20–30 ml of wash buffer (buffer A plus 20 mM imidazole), the protein was eluted with 4–6 ml of elution buffer (buffer A containing 500 mM imidazole). FAD (100 μM) was added to eluted proteins before storage at −80°C until further use. The affinity-purified proteins were further separated for use in in vitro studies by gel filtration chromatography using a Superdex 200 (or Superdex 75) 10/30 column connected to an ÄKTA-FPLC system (GE Healthcare) at 4°C in BAE (150 mM NaCl, 50 mM Tris/HCl, 1 mM EDTA, pH 7.4).