Computational protocol: Structural insights into chemokine CCL17 recognition by antibody M116

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Protocol publication

[…] Crystallization was carried out by the vapor-diffusion method at 20 °C using an Oryx4 robot (Douglas Instruments). The experiments were composed of equal volumes of protein and reservoir solution in a sitting drop format in 96-well Corning 3550 plates. The initial screening was performed with the PEGs kit (Qiagen) and in-house screens. The crystal hits were optimized by microseed matrix screening . The Fab crystals suitable for X-ray analysis were obtained from 18% PEG 3350 in 0.1 M HEPES, pH 7.5. The Fab-CCL17 complex was crystallized from 20% PEG 3350, 0.2 M K/Na tartrate at pH 7.4.For X-ray data collection, one crystal of each the Fab and the complex was soaked for a few seconds in the respective mother liquor supplemented with 20% glycerol, and flash-cooled in liquid nitrogen. Diffraction data were collected with a Pilatus 6 M detector at the Advanced Photon Source (Argonne, IL) with the exposures of 1 s per half-degree image and were processed with XDS . X-ray data statistics are given in .The structure of the Fab-CCL17 complex was determined by molecular replacement with Phaser using the structures of CCL17 (PDB entry 1nr4) and of human germline antibody 5-51/B3 (PDB entry 5i1l) as search models. The refined Fab structure was then used in structure determination of the unbound Fab. Both structures were refined with Refmac5 . Manual adjustments were performed using Coot . All crystallographic calculations were performed with the CCP4 suite of programs . Ramachandran statistics were calculated with PROCHECK . Figures were prepared with PyMol (Schrödinger). […]

Pipeline specifications

Software tools XDS, REFMAC5, Coot, CCP4, PROCHECK, PyMOL
Applications Small-angle scattering, Protein structure analysis
Diseases Asthma, Colitis, Ulcerative, Dermatitis, Atopic, Idiopathic Pulmonary Fibrosis