Computational protocol: Potent Dengue Virus Neutralization by a Therapeutic Antibody with Low Monovalent Affinity Requires Bivalent Engagement

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Protocol publication

[…] DENV-1 DIII (residues 293 to 399 of the E protein of strain 16007) was expressed in bacteria and re-folded oxidatively from isolated inclusion bodies as described previously . Fab fragments of E106 were prepared using immobilized papain resin according to the manufacturer's instructions (Pierce). MAb (5 to 10 mg) was digested for 18 hours at 37°C, and passed over a protein A agarose resin to remove Fc fragments and undigested MAb and purified on a S-75 size exclusion chromatography column equilibrated in 20 mM HEPES pH 7.4 and 150 mM NaCl. Antibody–antigen complexes were formed by mixing papain-generated, gel filtered E106 Fab fragments with DIII at a ratio of 1.2∶1 and crystallized by the hanging drop vapor diffusion method at a total protein concentration of 14 mg/mL in a solution of 22% PEG 6,000, and 0.1 M MES pH 5.0 (final pH 5.7). Crystals (in 1 µL crystallization drops) were cryoprotected by the addition of 0.2 µL aliquots of cryobuffer (in 23.5% PEG 6,000, 0.1 M MES pH 5.0, final pH 5.7, and 20% glycerol), then transferred to a fresh drop of cryobuffer and rapidly cooled in liquid nitrogen. X-ray diffraction data were collected at ALS beamline 4.2.2 (Lawrence Berkeley Laboratories) at a wavelength of 0.976 Å at 100 K with a CCD detector, and indexed and scaled in HKL2000 . The crystals diffracted to 2.45 Å resolution and belonged to the space group P212121 with unit cell dimensions of a = 82.7 Å, b = 91.8 Å, c = 92.6 Å, with one E106 Fab-DIII complex per asymmetric unit. Crystallographic phasing was obtained by molecular replacement using the program Phaser and the coordinates of DENV-1 DIII (Protein Data Bank (PDB) 3EGP) and the Fab fragment of CTM01 IgG (PDB 1AD9). Iterative model building and refinement was performed using Coot and Refmac and later Phenix . The final structure was refined to Rcryst = 18.9% and Rfree = 23.9%. The final model includes DENV-1 DIII amino acid residues 297 to 394, E106 heavy chain residues 1 to 214 (Chothia numbering), and light chain residues 1 to 213. The atomic coordinates and structure factors of E106 Fab bound to DENV-1 DIII (CSGID target number IDP00272) have been deposited in the Protein Data Bank (www.rcsb.org) under PDB accession number 4L5F. Structural figures were prepared using CCP4MG and Pymol (surface representation using 1.4 Å solvent probe) and where shown, spheres represent van der Waal radii, vdw * 1.1. […]

Pipeline specifications

Software tools Coot, PHENIX, CCP4mg, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Mus musculus
Diseases Infection, HIV Infections