Computational protocol: Iron Binding at Specific Sites within the Octameric HbpS Protects Streptomycetes from Iron-Mediated Oxidative Stress

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Protocol publication

[…] HbpS-D78XXD81 was crystallized at a concentration of 10 mg/ml using the sitting-drop method in a 96-well plate at 295 K. The crystallization condition contained 20% (w/v) polyethylene glycol 3350 and 0.2 M tri-potassium citrate. The crystal was frozen immediately prior to data collection directly in a stream of nitrogen at 100 K without additional cryo-protection. 80 degrees of diffraction data using an oscillation of 0.2 degrees per frame were collected on a Pilatus 6M detector mounted on the EMBL beamline P13 at PETRA III (DESY). The crystal belonged to the space group I422 and contained one molecule in the asymmetric unit. The biological octamer, as previously observed , is formed by crystallographic symmetry. The data were processed using XDS and XSCALE to 1.99 Å resolution ().The structure was solved by molecular replacement using the program Phaser in the Phenix interface . One monomer of the wild-type HbpS (PDB: 3FPV) was used as the search model. Refinement was carried out using the program phenix.refine and model building in Coot . The final model displays good geometry and R/Rfree factors of 0.183 and 0.234, respectively (). The structure factors and coordinates have been submitted to the PDB under the code 4BMW. […]

Pipeline specifications

Software tools XDS, PHENIX, Coot
Applications Small-angle scattering, Protein structure analysis
Chemicals Iron