Computational protocol: A variable DNA recognition site organization establishes the LiaR-mediated cell envelope stress response of enterococci to daptomycin

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Protocol publication

[…] Crystals of DBD LiaR suitable for data collection were obtained in 0.1 M Tris pH 8.6, 0.2 M LiSO4, 20% (w/v) PEG 3.350, 0.05% Tween-20, 10% glycerol, 10 mM praseodymium (III) acetate hydrate. The diffraction data sets were collected at Argonne National Laboratory's Advanced Photon Source (ANL APS) beamline 21-ID-F on a MarMosaic 225 CCD detector. The structure of LiaR(DBD) protein was solved by molecular replacement () using the DNA binding domains of beryllofluoride-activated VraR from Staphylococcus aureus (PDB code: 4IF4) as an initial search model. The solution from molecular replacement suggested two molecules in the asymmetric unit and a Matthews coefficient of 3.03 (59.5% solvent content) (). The initial model was submitted to phenix.autobuild and phenix.refine for automatic building and structure refinement (). Phenix.Xtriage () analysis was performed for initial data characterization. Data analysis suggested that pseudo-merohedral twining by the twin law, −h,l,k, was possible; however, no significant pseudotranslation was detected by Patterson analysis. Structural refinement was carried out in Phenix: phenix.refine () with the twin specific target function. The initial model was improved by iterative rebuilding using (2Fo − Fc) a map made with Coot (). The applicable pseudo-merohedral twin law, −h,l,k, was applied from the beginning and throughout refinement in Phenix.refine and the twin fraction reached a final value of 0.49. Water molecules were added using phenix.refine program and by manual inspection of 2Fo − Fc electron density maps. The structure of the LiaRDBD(D191N) protein was solved by molecular replacement () using the LiaRDBD structure as an initial search model. Structural refinement was carried out in Phenix: phenix.refine with the twin specific target function (−h,l,k, twin fraction is 0.49) (Supplemental Information). Ramachandran plots and root mean square deviations from ideality for bond angles and lengths for LiaRDBD and LiaR(DBD)D191N were determined using the structure validation program MolProbity (). Structure factors and final atomic coordinates have been deposited with the Protein Data Bank (entries: 4WSZ and 4WT0, respectively) […]

Pipeline specifications

Software tools PHENIX, Coot, MolProbity
Application Protein structure analysis
Organisms Enterococcus faecalis
Chemicals Daptomycin