Computational protocol: Structural and Biochemical Characterization of the Francisella tularensis Pathogenicity Regulator, Macrophage Locus Protein A (MglA)

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Protocol publication

[…] Crystals of MglA and selenomethionine-substituted MglA were grown using 2.1 M Malic acid, pH 7.0 as the crystallization solution. The structure was determined using multiple wavelength anomalous diffraction (MAD) from data collected on a selenomethionine-substituted MglA crystal. For the MAD experiment, X-ray intensity data were collected at three wavelengths (peak, inflection and remote) at ALS beamline 8.3.1 (). The images were integrated using Mosflm [] and scaled with SCALA [,]. The heavy atom substructure was obtained using SOLVE and phases and density modification carried out in CNS. Although the map was of excellent quality, the resolution was not sufficient to permit autotracing [,]. Hence, the structure was constructed manually using Coot []. The structure was refined with phenix.refine to final Rwork and Rfree values of 17.3% and 22.8%, respectively []. The final structure includes residues 1–201 and 1–197 for two MglA molecules in the crystallographic asymmetric unit (ASU), 2 malate molecules and 70 water molecules. []. Final refinement statistics are listed in . [...] A model of F. tularensis SspA was generated from its protein sequence using Phyre 2 []. Briefly, Phyre2 predicts a protein structure from its sequence using homology modelling. A profile for the protein is generated with PSI-blast, and then analyzed for secondary structure with PSI-pred. The success of the modelling is determined largely by how well the structure can be predicted with sequence alignments. Phyre2 is able to generate a reasonable model with a protein such as SspA, where several structures and homologous sequences are available. The resultant F. tularensis SspA model was then aligned in PyMOL onto the B subunit of the MglA dimer to generate the SspA•MglA heterodimer model. […]

Pipeline specifications

Software tools iMosflm, CCP4, CNS, Coot, PHENIX, Phyre, BLASTP, PSIPRED, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Francisella tularensis
Diseases Tularemia