Computational protocol: Human antibody 3E1 targets the HA stem region of H1N1 and H5N6 influenza A viruses

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Protocol publication

[…] To prepare the 3E1 Fab–HA complex, the purified 3E1 Fab and HA were mixed in a 2:1 molar ratio (Fab:monomeric HA, six Fabs per HA trimer) and incubated overnight at 4 °C, and the excess Fab was removed by size exclusion chromatography with a Superdex 200 10/300 column. Crystallization was performed using the hanging drop vapour diffusion method at 20 °C by mixing an equal volume (0.1 μl) of the protein solution (10 mg ml−1) and the reservoir solution. Crystals of the 3E1 Fab–CA09 HA complex were grown from the drop with the reservoir solution containing 0.1 M MES (pH 6.5) and 16% (v/v) PEG 550 MME over two months. Crystals of the 3E1 Fab–WA11 HA complex were grown from the drop with the reservoir solution containing 0.1 M Na HEPES (pH 7.5), 20% (v/v) PEG 400 and 0.1 M LiCl over 4 months. Before collecting the diffraction data, the crystals were cryoprotected in the reservoir solution supplemented with 25% (v/v) glycerol, and then flash cooled in liquid nitrogen. The diffraction data were collected at −175 °C at BL19U1 of the National Facility for Protein Science in Shanghai and BL17U of the Shanghai Synchrotron Radiation Facility, and processed with HKL2000 (ref. ).The structure of the 3E1 Fab–CA09 HA complex was solved by the molecular replacement (MR) method, as implemented in Phaser of Phenix using the monomeric CA09 HA (PDB code 3LZG) and the daclizumab Fab (PDB code 3NFS) as the search models. The structure of the 3E1 Fab–WA11 HA complex was solved using the 3E1 Fab–CA09 HA complex as the search model. Structural refinement was performed with Phenix and Refmac5 (ref. ), and the models were constructed using Coot. The stereochemistry and quality of the structural models were analysed using Molprobity, the PISA server and programs in the CCP4 suite and were validated using the wwPDB validation server ( All structures were generated using PyMOL ( The diffraction data and structural refinement statistics are summarized in . […]

Pipeline specifications

Software tools PHENIX, REFMAC5, Coot, MolProbity, CCP4, PyMOL
Databases wwPDB
Application Protein structure analysis
Organisms Viruses, Homo sapiens, Mus musculus