Computational protocol: Structure of LdtMt2, an l,d-transpeptidase from Mycobacterium tuberculosis

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Protocol publication

[…] X-ray data sets were collected to 1.45 Å resolution for the AB module and to 1.86 Å resolution for the BC module on beamline ID14-1 of the European Synchrotron Radiation Facility (ESRF), Grenoble, France. In addition, a data set was collected to 2.7 Å resolution from a crystal of the SeMet-substituted BC module on beamline ID29 at the ESRF at the Se edge at a wavelength of 0.97677 Å. All data sets were collected at 100 K. The X-ray data were processed and scaled using XDS (Kabsch, 2010; AB module) and MOSFLM (Leslie, 2006; BC module) and scaled with SCALA from the CCP4 suite (Winn et al., 2011).The crystals of the AB module belonged to space group C2, with unit-cell parameters a = 114.4, b = 27.6, c = 68.0 Å, β =114.1°, and contained one molecule in the asymmetric unit with a solvent content of 47.8%. Crystals of the BC module (native and selenomethionine-substituted) belonged to space group I212121, with unit-cell parameters a = 116.0, b = 121.4, c = 123.3 Å, and contained two molecules in the asymmetric unit with a solvent content of 68%. The statistics for all data sets are summarized in Table 1. [...] The structure of the BC module was solved by Se-SAD phasing using the data sets obtained from crystals of the SeMet-substituted protein. The Se sites were identified and refined from the peak data set using SHELX (Sheldrick, 2008). Of the 12 selenium sites in the asymmetric unit, 11 were found by SHELX, with CC values of 47.2% (CCall) and 30.1% (CCweak). An almost complete model was built by ARP/wARP (Mooij et al., 2009) using the SeMet peak data set. From this point, the 1.86 Å resolution native data set was used to complete the model by iterative rounds of manual model building using Coot (Emsley et al., 2010) and crystallographic refinement with REFMAC5 (Murshudov et al., 2011) applying local NCS symmetry and TLS. The final model contained two chains of the BC module comprising protein residues 149–408, 12 acetate ions, two Na+ ions and 480 water molecules. The final crystallographic R and R free values were 16.5% and 18.7%, respectively (Table 1).The structure of the AB module was solved by molecular replacement using Phaser (McCoy et al., 2007) with the B domain (149–250) derived from the BC module as the search model. Density modification using Parrot (Cowtan, 2010) was employed to improve the electron-density map, in particular for the unknown domain A at the N-­terminus. The A domain was built manually using Coot (Emsley et al., 2010) and refined to 1.45 Å resolution by cycles of restrained refinement using REFMAC5 (Murshudov et al., 2011). The final structure comprised amino-acid residues 57–250, two sulfate ions and 309 water molecules; the crystallographic R and R free values were 16.2% and 21.8%, respectively (Table 1).Both protein models were validated using Coot (Emsley et al., 2010) and MolProbity (Chen et al., 2010) to monitor the stereochemistry and model quality. A summary of the refinement statistics and model parameters is given in Table 1. Structural comparisons were carried out using the DALI algorithm (Holm & Rosenström, 2010; Holm & Park, 2000). Molecular contacts and interacting surfaces were analyzed using the PISA server (Krissinel & Henrick, 2007). Figures were produced using the program PyMOL ( The coordinates of the AB module and BC module have been deposited in the Protein Data Bank under accession codes 4hu2 and 4huc, respectively. […]

Pipeline specifications

Software tools XDS, iMosflm, CCP4, ARP/wARP, REFMAC5, MolProbity, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Mycobacterium tuberculosis
Diseases Tuberculosis