Computational protocol: Outward- and inward-facing structures of a putative bacterial transition-metal transporter with homology to ferroportin

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Protocol publication

[…] All diffraction data sets were collected at SPring-8 BL32XU, using a micro-focused X-ray beam, and processed using the programme XDS. For the phase determination, the data sets collected from the native crystal and the Hg-derivative crystal were analysed using the programs SHELXC and D (ref. ), and one Hg atom site was identified. Based on this information, the initial phases were calculated using the programme SHARP, by the single isomorphous replacement with anomalous scattering method. The initial model was built using the programme PHENIX AutoBuild. The model was further built manually using COOT and refined using PHENIX, and finally the structure was determined at 2.5 Å resolution. By utilizing this structure as the search model, the form I structure was determined by molecular replacement with the programme PHASER. The form II structure was determined by molecular replacement using the refined form I structure, with separate searches for the N lobe and the C lobe. For the model building and the refinement of both the form I and form II structures, COOT and PHENIX were used. The data collection and refinement statistics are summarized in . All molecular graphics were prepared using CueMol (http://www.cuemol.org/). [...] Based on the outward-facing structure of BbFPN, homology models of hFPN were generated using the programme MODELLER. The orientations of the residues around the metal-binding site were manually adjusted, using the programme COOT. The Fe2+ ion was placed at the position corresponding to the K+ in the BbFPN structure. The hepcidin–hFPN docking model was constructed manually, using COOT. […]

Pipeline specifications

Software tools XDS, SHELX, PHENIX, Coot, CueMol, MODELLER
Applications Small-angle scattering, Protein structure analysis
Diseases Anemia
Chemicals Iron