Efficient Recombinant Protein Production

All ectodomains were produced in 500-ml cultures of HEK293F cells grown in suspension using FreeStyle 293 expression medium (Life technologies) at 37°C in a humidified 8% CO₂ incubator rotating at 130 r.p.m., as previously reported^{18 (link)}. The culture was transfected using 293fectin (ThermoFisher) with cells grown to a density of 10⁶ cells per ml and cultivated for 3 d. The supernatant was collected and cells were resuspended for another 3 d, yielding two collections. Clarified supernatants were purified using a 5-ml Cobalt affinity column (Takara). Purified protein was concentrated, and flash frozen in a buffer containing 50 mM Tris pH 8.0 and 150 mM NaCl before cryo-EM analysis.