Computational protocol: Host Determinant Residue Lysine 627 Lies on the Surface of a Discrete, Folded Domain of Influenza Virus Polymerase PB2 Subunit

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Protocol publication

[…] Crystals of the 627-domain (residues 538–693) with Lys627 (native and selenomethionine labelled), the 627-domain with Glu627 (native) and the 627-NLS-domain (native, residues 538–753) were measured at the European Synchrotron Radiation Facility (ESRF). gives all data collection and refinement statistics. All crystals have one molecule in the asymmetric unit. All data were integrated with XDS and analysed using the CCP4i package . The structure of the 627-domain with Lys627 was solved by the SAD method using AUTOSHARP which found 5 selenium positions. ARP/wARP was used for automatic model building. The structure of the K627E mutant was obtained by refinement. The double domain structure was solved by molecular replacement using PHASER and, as search models, the 627-domain and the NLS-domain from the complex with human importin α5 (PDB id: 2JDQ). All refinements were performed with REFMAC with added hydrogen atoms. For the very high resolution native 627-domain and K627E structures individual atomic anisotropic B-factors were refined. In the 627-domain alone structures, the 640–644 loop is disordered and the 609–610 loop has multiple conformations; both regions are well ordered in the 627-NLS-domain structure. The linker region 678–685 is poorly ordered in the 627-NLS-domain structure, but residual discontinuous density unambiguously defines which 627-domain is connected to which NLS-domain in the crystallographic asymmetric unit. Diffraction data for the 627-domain alone extend to very high resolution (1.1 Å for the native Lys627 data). Paradoxically the highest resolution data does not yield the most complete model; for example in the Lys627 native structure there is no electron density for the extended C-terminal tail of the 627-domain (residues 676–693), whereas this is perfectly ordered in the SeMet data and mostly ordered in the Glu627 data. In the latter two structures many multiple conformations can be modelled (). According to MOLPROBITY all structures have excellent geometry (http://molprobity.biochem.duke.edu/). […]

Pipeline specifications

Software tools XDS, ARP/wARP, MolProbity
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma, Homo sapiens
Diseases Influenza, Human