Computational protocol: Acid Stability of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Pathogenicity

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Protocol publication

[…] HA protein crystals were grown by the hanging-drop vapor diffusion method at 18°C. MP HA crystallized in a well solution of 23% PEG 3350 and 0.1 M Tris-HCl, pH 8.5. From HP HA, two crystal forms were obtained in the same crystallization conditions (1.62 M ammonium sulphate, 0.1 M sodium cacodylate, pH 6.6). Crystals were transferred to a well solution containing 25% glycerol (MP HA) or 25% ethylene glycol (HP HA) for 1–2 minutes before freezing in liquid nitrogen. Diffraction data were collected at cryogenic temperature at X-ray wavelength 1.00 Å from the Southeastern Regional Collaborative Access Team's 22-ID and 22-BM beamlines at the Advanced Photon Source (Argonne National Laboratory, Chicago, IL). Data processing and reduction was completed by using HKL-2000 software .HA ectodomain structures were determined by molecular replacement using the program Phaser . From HP HA crystal form 1, a solution was obtained by using a single HA protomer from the crystal structure of the HA from H5N1 A/Vietnam/1203/2004 (PDB entry 2FK0). For MP HA, the HP HA crystal form 1 structure was used as a molecular replacement model. For HP HA crystal form 2, the best molecular replacement solution was obtained by using a single HA protomer from MP HA's crystal structure. Model building was performed by using Coot followed by iterative rounds of simulated annealing using Phenix and restrained refinement using the CCP4 software suite's REFMAC5 . Refinement was monitored by following the Rfree value calculated for a random subset (5%) of reflections omitted from refinement. The final models were validated by using MolProbity and are numbered according to H3 numbering based on the crystal structure of A/Vietnam/1203/2004 H5 HA (PDB entry 2FK0). We used the H3 numbering scheme in this manuscript, which differs from the H5 numbering that was used previously .After simulated annealing of HP HA crystal form 2, the electron density for the region of the stalk domain that is closest to the viral membrane was very poor due to irregular crystal packing within this region. The structural model of HP HA crystal form 2 was guided by B-factors: residues with B-factors higher than 90 were not included in the model. The final model of HP HA crystal form 2 contains HA1 residues 43–312 and HA2 residues 59–101 (H3 numbering). [...] HA protein sequences published between 1996 and 2011 (as of May 2011) were obtained from NCBI's Influenza Virus Resource database (http://www.ncbi.nlm.nih.gov/genomes/FLU/). Laboratory sequences or sequences that did not cover the amino-acid positions of interest in this study were excluded. Sequences were aligned by using the ClustalW tool included in BioEdit v7.0.9 . The frequencies of amino-acid residues were calculated for HA1 positions 104, 107, 109, and 115 and HA2 positions 73, 69, and 76 (in H3 numbering). […]

Pipeline specifications

Software tools Clustal W, BioEdit
Databases Influenza Virus Resource
Application Protein sequence analysis
Organisms Gallus gallus, Homo sapiens, Dipturus trachyderma
Chemicals Amino Acids