Computational protocol: A recurring motif for antibody recognition of the receptor-binding site of influenza hemagglutinin

Similar protocols

Protocol publication

[…] An excess of purified, recombinant 8F8 Fab was mixed with H2 HA (L226Q, S228G mutant of A/Japan/305+/1957) and incubated overnight at room temperature to allow complex formation. The optimal ratio was pre-determined by titration of Fab to HA as assayed by gel-shift using Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) (Invitrogen). The complex was purified from excess Fab by gel filtration in 20 mM Tris pH 8.0, 50 mM NaCl, and concentrated to 6.2 mg/mL for crystallization. 8F8-H2 complex crystals were grown at 22.5°C in sitting drops by vapor diffusion against a reservoir containing 11% PEG4000, 0.1 M Tris, pH 8.5 and 0.1 M MgCl2. The crystals were cryoprotected by stepwise addition (5% each) of ethylene glycol, up to a final concentration of 25%, and then flash cooled in liquid nitrogen. Diffraction data were collected at 100K on the General Medicine and Cancer Institutes Collaborative Access Team (GM/CA-CAT) 23ID-B beamline (wavelength: 1.0332 Å) at the Advanced Photon Source at the Argonne National Laboratory and processed with HKL2000 . Initial phases were determined by molecular replacement using Phaser . Refinement was carried out in Phenix , alternating with manual rebuilding and adjustment in COOT . Final refinement statistics are summarized in . In the final model, 92.3% of the residues were in favored regions of the Ramachandran plot, with 7.3% in additional allowed regions. […]

Pipeline specifications

Software tools PHENIX, Coot
Application Protein structure analysis
Organisms Viruses
Diseases Infection, HIV Infections
Chemicals Tyrosine, N-Acetylneuraminic Acid