Computational protocol: Structural Insights into the Membrane Fusion MechanismMediated by Influenza Virus Hemagglutinin

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Protocol publication

[…] HA2 residues 31–181 of influenza B/Yamagata/73 were cloned into vector pET-45b with an N-terminal six-histidine tag and expressed in Escherichia coli Rosetta 2(DE3)pLysS (Novagen). Expressed HA2 was purified using cobalt resin (Thermo Scientific) and size-exclusion chromatography (Superdex 200 10/300 GL, GE Healthcare). Purified HA2 was concentrated to 10 mg/mL in 10 mM Hepes (pH 7.6), and crystals were grown at 290 K by the hanging drop method in a reservoir solution of 0.1 M ammonium citrate and 13.5% PEG 3350 (pH 7.0). The diffraction data were collected using the 14-BMC beamline (BioCARs) at the Advanced Photon Source (Chicago, IL), indexed and integrated by using MOSFLM,, scaled by SCALA, and truncated to structure factor amplitude by TRUNCATE in CCP4. Five percent of unique reflections omitted from refinement were used as the test set for calculating the Rfree values.The long helix from the postfusion structure of influenza A/H3N2 virus HA2 [Protein Data Bank (PDB) entry 1QU1], including residues 37–105, was pruned to Cβ atoms by CHAINSAW in the CCP4 suite and served as the search model for molecular replacement by the AutoMR module implemented in PHENIX. There is one polypeptide chain of HA2 in the asymmetric unit. The biological HA2 trimer was generated by symmetry operation. The resulting σ-weighted 2Fo – Fc map showed clear densities for the rest of the protein that were built into the density by COOT. The model was refined by REFMAC5 in CCP4 or the Refinement module in PHENIX. Figures for structural snapshots were generated by using Pymol. […]

Pipeline specifications

Software tools iMosflm, CCP4, PHENIX, Coot, REFMAC5, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Influenza B virus
Diseases Hepatitis B, Influenza, Human