Computational protocol: Unveiling structural and functional divergences of bacterial tRNA dihydrouridine synthases: perspectives on the evolution scenario

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Protocol publication

[…] Initial crystals of DusB were obtained by mixing 1 μl of protein at 10 mg/ml in 50 mM Tris pH 8 and 150 mM NaCl with 1 μl of 1.6 M lithium sulfate and 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5 by vapor diffusion at 292 K. Due to poor diffraction quality, crystallization assays at 277 K were carried out and led to higher diffraction properties. Crystals were cryoprotected with a solution consisting of the reservoir supplemented with 15% v/v glycerol and flash frozen. X-ray diffraction data were collected at 100 K on a single crystal at PROXIMA-2a beamline at the SOLEIL synchrotron (Saint-Aubin, France) using an Eiger X-9M. Data were indexed and processed using XDS and corrected for anisotropy with the STARANISO server (staraniso.globalphasing.org). Structure solution was obtained by molecular replacement with Phaser using as a search template 2 monomers of E. coli DusC (PDB code: 4BFA) that shares 29.4% sequence identity with DusB. A clear solution was obtained (TFZ = 12.8; LLG = 154 723). Refinement was done with autoBUSTER using NCS and alternating with manual building in Coot. The entire HD was rebuilt manually into the density. One TLS group per chain was used at the end of the refinement. [...] Small-angle X-ray scattering (SAXS) experiments were performed at the SWING beamline at the SOLEIL synchrotron (Saint-Aubin, France) using an online HPLC. All scattering intensities were collected on the elution peaks after injection on a BioSEC-3 column (Agilent) equilibrated in 25 mM HEPES pH 8, 500 mM NaCl and 5% glycerol. Data were processed using FOXTROT (). The radius of gyration (Rg) was calculated by the Guinier approximation.Coral or bunch were used to construct the 19 missing residues (44–62) not observed in the X-ray structure (,). Ten independent runs in each case were averaged and compared to the experimental curve with crysol. Both approaches led to similar models. […]

Pipeline specifications

Software tools XDS, Coot, ATSAS, CRYSOL
Applications Small-angle scattering, Protein structure analysis
Organisms Bacteria, Escherichia coli
Diseases Neoplasms
Chemicals Uridine