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

Similar protocols

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 ( 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