Crystallization of the Atg12–Atg5 conjugate bound to Atg16 by the free-interface diffusion method

Autophagy mediates the bulk degradation of cytoplasmic components in lysosomes/vacuoles. Five autophagy-related (Atg) proteins are involved in a ubiquitin-like protein conjugation system. Atg12 is conjugated to its sole target, Atg5, by two enzymes, Atg7 and Atg10. The Atg12–Atg5 conjugates form a multimeric complex with Atg16. Formation of the Atg12–Atg5–Atg16 ternary complex is crucial for the functions of these proteins on autophagy. Here, the expression, purification and crystallization of the Atg12–Atg5 conjugate bound to the N-terminal region of Atg16 (Atg16N) are reported. The Atg12–Atg5 conjugates were formed by co-expressing Atg5, Atg7, Atg10 and Atg12 in Eschericia coli. The Atg12–Atg5–Atg16N ternary complex was formed by mixing purified Atg12–Atg5 conjugates and Atg16N, and was further purified by gel-filtration chromatography. Crystallization screening was performed by the free-interface diffusion method. Using obtained microcrystals as seeds, large crystals for diffraction data collection were obtained by the sitting-drop vapour-diffusion method. The crystal contained one ternary complex per asymmetric unit, and diffracted to 2.6 Å resolution.