Computational protocol: Specificity Determinants for Lysine Incorporation in Staphylococcus aureus Peptidoglycan as Revealed by the Structure of a MurE Enzyme Ternary Complex*

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Protocol publication

[…] X-ray data on the ADP and UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys liganded crystals were collected on the IO2 beamline at the Diamond Light Source synchrotron (Didcot, UK) using an ADSC Q315 CCD detector. All data were indexed, integrated, and scaled using the XDS package (). Subsequent data handling was carried out using the CCP4 software package (). Molecular replacement was carried out using the coordinates of E. coli MurE (MurEEc; Protein Data Bank (PDB) code 1E8C ()) as a search model with the PHASER program (). Refinement of the structure was carried out by alternate cycles of REFMAC () and manual rebuilding in O (). Water molecules were added to the atomic model automatically by Arp/wARP ().Data on the crystal liganded with UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys only were collected using a Xenocs GeniX3D Cu HF (High Flux) microbeam x-ray generator with a Mar345 imaging plate. The data were processed with iMOSFLM () and reduced with SCALA from the CCP4 suite (). The MurESa·UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys complex was solved by molecular replacement using MolRep () and the protein atomic coordinates of the product complex. The structure was refined using iterative cycles of REFMAC () and model building/solvent addition with COOT (). Translation/libration/screw motion, as determined by the TLSMD web server (), was included in refinement. For both structures, the space group was C2, and there was one molecule in the asymmetric unit. A summary of the data collection and refinement statistics is given in . Figures were drawn using PyMOL (). […]

Pipeline specifications

Software tools XDS, CCP4, ARP/wARP, iMosflm, Molrep, Coot, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma, Staphylococcus aureus
Chemicals Adenosine Diphosphate, Adenosine Triphosphate, Amino Acids, Uridine Diphosphate