Computational protocol: Structural characterisation of the virulence-associated protein VapG from the horse pathogen Rhodococcus equi

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Protocol publication

[…] Crystals were obtained using a protein solution consisting of VapG at 30 mg ml−1 in 10 mM Tris–HCl (pH 7.5), 10 mM NaCl. The crystals grew at 18 °C by hanging drop vapour diffusion against a reservoir solution containing 0.3 M potassium chloride, 20% PEG 3350, originating from the Peg/Ion screen (Hampton Research). A crystal of approximate dimensions 400 μm × 400 μm × 400 μm was vitrified in cryoprotectant solution (30% glycerol, 70% reservoir) and X-ray diffraction data were collected at 100 K at the Diamond Light Source experimental station I02. Data were integrated using XDS () and scaled/merged with Aimless (). The structure of VapG was solved by molecular replacement with the program MOLREP () using a family-related structure VapD (PDB code 4csb, ) as a search model, this sharing 60% amino acid sequence identity with VapG (B). Two solutions were found equating to two molecules of VapG per asymmetric unit. The resulting model was refined using maximum likelihood methods implemented in REFMAC5 () with 5% of the total data excluded from the refinement for the purpose of R free calculations. This procedure was interspersed with manual corrections to the model using COOT () in conjunction with 2Fo − Fc and Fo − Fc electron density maps.During refinement, elongated peaks of positive difference electron density appeared in two independent locations in the asymmetric unit. These peaks had the appearance of additional protein chains or peptides bound between the protein molecules: in one location a sequence of three histidines was built and successfully refined; in the other the sequence ALAA was built. It was concluded that these elements formed parts of the C-terminal affinity tags from the two VapG molecules, with the ALAA corresponding to the tag sequence KLAA in one molecule and AALE in its symmetry mate. Another striking feature in the electron density maps was a strong metal ion peak located at the centre of a cluster of main chain and side chains oxygen donors from residues 107 to 114 of molecule A. The coordination distances ranged from 2.7 to 3.1 Å which was consistent with a potassium ion (). Although other metal ion candidates (calcium and magnesium) were tested out in refinement, potassium was the only one which refined satisfactorily. Furthermore, X-ray absorption analysis of a single crystal revealed no other metal ions present apart from potassium, which was a crystallisation component. Refinement statistics and structural information are listed in . [...] During analysis of the structure, multiple sequence alignment was performed using CLUSTAL-W () and displayed using ESPript 3.0 (). Three dimensional structural alignments were made using secondary-structure matching () implemented in COOT (). All figures were generated using CCP4mg (). […]

Pipeline specifications

Software tools XDS, CCP4, Molrep, REFMAC5, Coot, Clustal W, CCP4mg
Applications Small-angle scattering, Protein structure analysis
Organisms Rhodococcus hoagii, Equus caballus
Diseases Pneumonia