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Updated: Jul 5, 2026
ABCG5/G8 Crystallization in a Lipidic Bicelle Environment for X-Ray Crystallography
Published on: August 25, 2023
Crystallization of the Atg12-Atg5 conjugate bound to Atg16 by the free-interface diffusion method
Nobuo N Noda1, Yuko Fujioka, Yoshinori Ohsumi
1Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, N-21, W-11, Kita-ku, Sapporo 001-0021, Japan.
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.
Insights
Researchers purified and crystallized the Atg12-Atg5-Atg16N complex, crucial for autophagy. This structural study provides insights into the ubiquitin-like conjugation system essential for cellular degradation processes.
Area of Science:
- Cell Biology
- Molecular Biology
- Structural Biology
Background:
- Autophagy is a fundamental cellular process for degrading cytoplasmic components.
- Autophagy relies on a ubiquitin-like protein conjugation system involving autophagy-related (Atg) proteins.
- The Atg12-Atg5 conjugate, complexed with Atg16, is essential for autophagy function.
Purpose of the Study:
- To report the expression, purification, and crystallization of the Atg12-Atg5 conjugate bound to Atg16N.
- To obtain a high-resolution structure of the Atg12-Atg5-Atg16N ternary complex.
- To facilitate structural studies of the autophagy machinery.
Main Methods:
- Co-expression of Atg5, Atg7, Atg10, and Atg12 in E. coli to form Atg12-Atg5 conjugates.
- Formation and purification of the Atg12-Atg5-Atg16N ternary complex using gel-filtration chromatography.
- Crystallization using free-interface diffusion and sitting-drop vapour-diffusion methods, with microcrystals as seeds.
Main Results:
- Successfully expressed and purified the Atg12-Atg5 conjugate and Atg16N.
- Assembled and purified the functional Atg12-Atg5-Atg16N ternary complex.
- Obtained crystals of the ternary complex that diffracted to 2.6 Å resolution, containing one complex per asymmetric unit.
Conclusions:
- The study successfully produced a crystallizable Atg12-Atg5-Atg16N ternary complex.
- The obtained crystal structure will enable detailed structural analysis of this key autophagy complex.
- This work provides a foundation for understanding the molecular mechanisms of autophagy regulation.
