title: Crystallization of the Atg12-Atg5 conjugate bound to Atg16 by the free-interface diffusion method.
abstract: 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 A resolution.
authors.affiliation: Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, N-21, W-11, Kita-ku, Sapporo 001-0021, Japan.
authors.forename: Nobuo N
authors.initials: NN
authors.lastname: Noda
authors.forename: Yuko
authors.initials: Y
authors.lastname: Fujioka
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Ohsumi
authors.forename: Fuyuhiko
authors.initials: F
authors.lastname: Inagaki
mesh_terms: D000071192:Autophagy-Related Protein 12; D003460:Crystallization; D004058:Diffusion; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D014961:X-Ray Diffraction
chemical_list: C491969:ATG12 protein, S cerevisiae; D000071192:Autophagy-Related Protein 12; D029701:Saccharomyces cerevisiae Proteins