Computational protocol: A Multi-Step Process of Viral Adaptation to a Mutagenic Nucleoside Analogue by Modulation of Transition Types Leads to Extinction-Escape

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Protocol publication

[…] Four different data sets were collected at 100 K: 3D(P44S) (2.2 Å), 3D(P169S) (2.6 Å), 3D(P44, M296I)-RNA (2.6 Å) and 3D(SSI)-RNA (2.5 Å), using synchrotron radiation at the ESRF beamlines ID14 EH1 and EH2 (λ = 0.93 Å). All data were processed and reduced using DENZO/SCALEPACK package ().The initial maps for the 3D(P44S) and 3D(P169S) (tetragonal crystals) were obtained after a rigid body fitting of the coordinates of isolated 3D protein that was crystallized in the tetragonal p42212 space group (PDB:1U09) to the new unit cells, using the program REFMAC (CCP4). Initial maps for the 3D(P44, M296I)-RNA and 3D(SSI)-RNA complexes (P3221 crystals) were obtained following the same procedure but using the trigonal P3221 coordinates of 3D (PDB:1WNE) as starting model (). In the four structures the 2|Fo|-|Fc| and |Fo|-|Fc| difference maps clearly allowed the re-positioning of the mutated residues and surrounding regions and, in the trigonal structures, these maps showed the presence of extra densities corresponding to the RNA template-primers. However, the tetragonal crystals, 3D(P44S) and 3D(P169S), did not contain RNA despite using the same incubation and co-crystallization conditions as in 3D(P44, M296I)-RNA and 3D(SSI)-RNA complexes that crystallized in the P3221 space group. Several cycles of automatic refinement, performed with program REFMAC, were alternated with manual model rebuilding using the graphic programs TURBO and Coot . The statistics of the refinement for the four complexes are summarized in . […]

Pipeline specifications

Software tools CCP4, Coot
Application Protein structure analysis
Diseases Foot-and-Mouth Disease
Chemicals Amino Acids, Nucleosides, Nucleotides, Purines, Ribavirin