Crystallization and structure determination
The complex containing ATG5E122D and the N-terminal domain of ATG16L1 (residues 1–69) was expressed in Hi5 insect cells, and purified by nickel affinity, ion exchange, and size exclusion chromatography into a final buffer of 20 mM Tris, pH 8.5, 50 mM NaCl, 10 mM DTT. The complex was concentrated to 18.5 mg/ml, aliquoted, flash-frozen and stored at -80°C until further use. Crystals were grown by the hanging drop vapor diffusion method by mixing purified protein 1:1 with reservoir solutions of 37.5 mM MES, pH 5.2–5.8, 0.2 M sodium tartrate, and 11–13% polyethylene glycol 3350. Final crystals were obtained by micro-seeding with reservoir solution of 40 mM MES, pH 5.5, 0.2 M sodium tartrate, 8.5% PEG3350, 10 mM DTT. Crystals were cryoprotected in reservoir solution supplemented with 25% xylitol, and flash frozen in liquid nitrogen prior to data collection. Diffraction data were processed with XDS. The structure was determined by molecular replacement using Phaser (McCoy et al., 2007) with the structure of the WT ATG5-ATG16L1 (1–69) (PDB: 4TQ0) complex as a search model (Kim et al., 2015a). Model construction and rebuilding were performed using Coot (Emsley et al., 2010). The structure was refined using Phenix (Adams et al., 2010). Diffraction data and refinement statistics are provided in Table 1.