Crystallization and structure determination

Z34C peptide was added to recombinant heterodimeric Fc at a 2.2:1 (Fc dimer) molar ratio. Following a 5 h incubation at room temperature, the sample was concentrated to 13.7 mg/mL in buffer containing 50 mM NaCl, 20 mM Tris, pH 8.0. Crystallization experiments employing the sitting drop vapor diffusion method were performed at room temperature using a Mosquito liquid handling robot (TTP Labtech, Ltd). 300 nL drops comprising protein stock and reservoir solution mixed at a 1:1 (v/v) ratio were set up in Corning 3550 plates. Diffraction quality crystals were grown at 20°C and were obtained with a reservoir solution comprising 17% (w/v) PEG 3350, 0.2 M LiCl, 0.1 M Tris, pH 8.0. Crystals were cryo-protected with reservoir solution supplemented with 20% (v/v) glycerol and flash frozen in liquid nitrogen prior to data collection. Diffraction data were collected at the Advanced Photon Source IMCA-CAT beamline 17-ID equipped with a Dectris Pilatus 6M pixel array detector and processed to 2.7 Å with the program XDS.⁴⁸ Initial phases were determined by molecular replacement with the program Phaser ⁴⁹ using a crystal structure of mouse IgG2b Fc (PDB ID 2RGS ⁵⁰) as a search model. The structure of Z34C from PDB 1L6X ³⁸ was subsequently manually docked into available 2Fo-Fc and Fo-Fc electron density followed by additional rounds of rebuilding and refinement using the programs Coot ⁵¹ and Phenix, ⁵² respectively. Data collection and final refinement statistics are given in Table 3. Analysis of contacts was performed using the program NCONT as implemented in the CCP4 suite of crystallographic programs ⁵³ using a cutoff distance of 5 Å. For comparison to other structures, alignments with performed with either the program LSQMAN⁵⁴ or PyMol (www.schrodinger.com/pymol). Figures were generated with PyMol.

Crystal data, X-ray data, and refinement statistics.