Computational protocol: In vitro evolution of an influenza broadly neutralizing antibody is modulated by hemagglutinin receptor specificity

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Protocol publication

[…] HA1 (H3 numbering: residues 43–309) from A/Hong Kong/1/1968 (HK68/H3) was expressed in insect cells as described and purified by Ni-NTA Superflow (Qiagen) and subsequently by size exclusion chromatography on a Hiload 16/90 Superdex 200 column (GE Healthcare) in 20 mM Tris pH 8.0, 150 mM NaCl, and 0.02% NaN3. The C05 variant VPGSGW was incubated with HK68/H3 HA1 in a molar ratio of 1.5:1 overnight at 4 °C. The VPGSGW-HK68/H3 HA1 complex was purified by size exclusion chromatography on a Hiload 16/90 Superdex 200 column (GE Healthcare) in 20 mM Tris pH 8.0, 150 mM NaCl, and 0.02% NaN3 and concentrated to ∼10 mg ml−1 in 10 mM Tris pH 8.0, 50 mM NaCl, and 0.02% NaN3. Crystal screening was carried out using our high-throughput, robotic CrystalMation system (Rigaku, Carlsbad, CA) at TSRI. Further optimization was based on the initial hit using the sitting drop vapour diffusion method with 500 μl reservoir solution containing 0.1 M sodium citrate pH 5.5 and 9% PEG 8000. Drops consisting 0.8 μl protein+0.8 μl precipitant were set up at 20 °C and crystals appeared within a week. The resulting crystals were cryoprotected by soaking in well solution supplemented with 15% PEG 400, flash cooled, and stored in liquid nitrogen until data collection.Diffraction data for the VPGSGW-HK68/H3 HA1 complex were collected at the Stanford Synchrotron Radiation Lightsource beamline 12-2. The data were indexed in space group P43, and integrated and scaled using HKL2000 (HKL Research, Charlottesville, VA). The structure was solved by molecular replacement at 1.97 Å resolution using Phaser with PDB 4FP8 (ref. ) as the molecular replacement model, modelled using Coot, and refined using Refmac5 (ref. ). Ramachandran statistics were calculated using MolProbity. [...] HA0 from A/Solomon Islands/3/2006 (SI06/H1) was treated with trypsin (New England Biolabs) to remove the C-terminal tags and cleave to produce mature HA. The trypsin-digested HA was then purified by size exclusion chromatography. VPGSGW was incubated with the purified HA in a molar ratio of 4.5:1 overnight. The VPGSGW-HA complex was purified by size exclusion chromatography. CR9114 Fab was then incubated with the purified VPGSGW-HA complex in a molar ratio of 4.5:1. Of note, additional VPGSGW was not supplied for this incubation and may explain the low occupancy of VPGSGW in the negative-stain data (). The CR9114-VPGSGW-HA complex was purified by size exclusion chromatography. All size exclusion chromatography were performed on a Hiload 16/90 Superdex 200 column (GE Healthcare) in 20 mM Tris pH 8.0, 150 mM NaCl, and 0.02% NaN3. The purified CR9114-VPGSGW-HA complex was added onto 400 mesh carbon coated copper grids with 2% uranyl formate. The grid was imaged on a Tecnai Spirit with camera at ∼1.5 μm defocus. The micrographs were collected using the program Leginon, processed using Appion, and particles were selected using DogPicker. Particles were aligned into two-dimensional (2D) classes and EMAN2 (ref. ) was used to generate an initial model. Using the model along with a clean stack of particles, which contained 18,874 particles at 2.05 Å per pixel, a final 3D reconstruction was produced at resolution of 11 Å (FSC=0.5). EMD maps or PDB models were docked into the density using Chimera. […]

Pipeline specifications

Software tools Coot, REFMAC5, MolProbity, Leginon, Appion, EMAN
Applications cryo-EM, Protein structure analysis
Organisms Homo sapiens, Saccharomyces cerevisiae