Computational protocol: Structural basis for therapeutic inhibition of influenza A polymerase PB2 subunit

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Protocol publication

[…] Crystals of PB2long were grown by hanging-drop vapor diffusion at 4 °C. The precipitant well solution consisted of 0.1 M MES pH 5.77, and 13.18% PEG 20,000. Crystals of the VX-787 complex were produced by soaking apo crystals in reservoir solution containing VX-787 at 1 mM final concentration. Crystals were cryo-protected by a harvesting solution containing reservoir solution supplemented by 20% glycerol and flash-cooled in liquid nitrogen for data collection. Datasets were collected under cryogenic conditions (100 K) at the Advanced Photo Source (APS) beamline 17-ID and Advanced Light Source (ALS) beamline 5.0.2. All data were processed using autoPROC, employing XDS for data integration and AIMLESS for scaling. Molecular replacement were carried out using PHASER with coordinates from PDB 2JDQ and 2VQZ representing the C-terminal domain and cap-binding domain, respectively. Iterative cycles of refinement and manual model building were carried out with PHENIX and COOT, respectively, until the models converged to acceptable levels of R factors and stereochemistry. […]

Pipeline specifications

Software tools XDS, CCP4, PHENIX, Coot
Applications Small-angle scattering, Protein structure analysis
Organisms Influenza A virus