Computational protocol: Cell-free synthesis of functional antibody fragments to provide a structural basis for antibody–antigen interaction

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Protocol publication

[…] Crystals of the 059-152-Fv•EGFR-ECD complex were obtained by using the sitting drop vapor diffusion method from a drop containing equal volumes of 059-152-Fv•EGFR-ECD solution and a reservoir solution [0.1 M MES buffer (pH 6.0) containing 0.1 M Zn(OAc)2 and 6% (w/v) PEG8000]. The crystals were immersed stepwise in reservoir solutions containing increasing concentrations of glycerol [15%, 20%, 25%, and 30% (v/v)]. The crystals were then flash cooled in liquid nitrogen. X-ray diffraction data were collected at the Photon Factory beamline AR-NE3A (Ibaraki, Japan). The dataset was processed by using HKL-2000 software []. The initial phase was determined with molecular replacement method using the software Phaser []. As a search model, the crystal structures of the extracellular domain of human EGFR-ECD [Protein Data Bank (PDB) ID code: 3NJP] and the Fv region of anti-EGFR Fab (PDB ID code: 3B2U) were used. The model was refined with the software Coot and Phenix [, ]. […]

Pipeline specifications

Software tools HKL-2000, Coot, PHENIX
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens
Chemicals Amino Acids, Hydrogen