Computational protocol: Methylation at position 32 of tRNA catalyzed by TrmJ alters oxidative stress response in Pseudomonas aeruginosa

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Protocol publication

[…] Prior to data collection, crystals were briefly soaked in their respective precipitating solution supplemented with 20% (v/v) glycerol and rapidly frozen in liquid nitrogen. X-ray diffraction data were collected at the PXIII beamline at SLS (Villigen, Switzerland) for the free TrmJ–NTD crystal (form I). Free TrmJ–NTD crystal (form II) and the complex with sinefungin were collected at Taiwan Light source NSRRC, beamline 13B1.Diffraction intensities were integrated with XDS (), scaled, merged and truncated with SCALA/TRUNCATE (). The structures of TrmJ–NTD was determined by molecular replacement using the CCP4 software () with the structure from E. coli (PDB code: 4CND) as search probe. The TrmJ protein from P. aeruginosa and E. coli share 53% overall amino acid sequence identity. The structure of TrmJ–NTD from P. aeruginosa was built iteratively at the computer graphics using COOT () and refined using Autobuster (). The geometrical parameters for sinefungin were generated using coordinates 4R8S from the PDB (www.rcsb.org). Buried solvent accessible surface areas upon dimer formation were calculated using the PDBePISA server (www.ebi.ac.uk/pdbe/pisa/) and structure comparison was performed with the DALI server (http://ekhidna.biocenter.helsinki.fi/dali_server/start). The quality of the structures was assessed using the MOLPROBITY server (molprobity.biochem.duke.edu/) and figures were generated using the Pymol software (Schrodinger). Data collection and structure refinement parameters are summarized in Table . The atomic coordinates and structure factors are deposited with the Protein Data Bank under accession codes 5GMC and 5GMB for the free TrmJ–NTD protein, and 5GM8 for TrmJ–NTD bound to sinefungin. […]

Pipeline specifications

Software tools XDS, CCP4, Coot, DALI, MolProbity, PyMOL
Databases PDBe
Applications Small-angle scattering, Protein structure analysis
Organisms Pseudomonas aeruginosa, Escherichia coli