The complex between NRTN and GFRα2 was formed by combining the proteins and incubating them for 3 hours at 4°C. Gel filtration was carried out on the mixture using a Superdex 75 column [50 mM Hepes (pH 7.5), 300 mM NaCl, and 10% glycerol] to separate monomeric proteins from the NRTN-GFRα2 complexes. Peak fractions were combined and added to human RET at a molar ratio of 1:2 in the presence of 20 mM CaCl2. Proteins were incubated for 3 to 16 hours at 4°C and purified via SEC (S200) in 20 mM Hepes (pH 8), 100 mM NaCl, and 1 mM CaCl2. Protein complexes were concentrated to 15 to 30 mg/ml, aliquoted, flash-frozen, and stored at −80°C.

Samples for cryo-EM were not concentrated, but the peak fraction (at ~1 mg/ml) was instantly used for grid preparation.

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