Computational protocol: Additional phase information from UV damage of selenomethionine labelled proteins

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Protocol publication

[…] All data were indexed and integrated using XDS (Kabsch, 2010) and scaled using XSCALE (Kabsch, 2010). Scaled dataset files were converted to SCALEPACK format with the software tool XDS2SCA (Ravelli, unpublished). SHELXC (Sheldrick, 2010) was used to prepare the input files for SHELXD (Schneider & Sheldrick, 2002) and to analyze the anomalous and the isomorphous signal of the collected data. The resolution for UV-RIPAS phasing was chosen such that 〈ΔF/σ(ΔF)〉 was greater that 1.5. F A values were calculated using the MAD and the SIRAS options in SHELXC. SHELXD was used to locate the substructure using the two-wavelength MAD and the SIRAS protocols. In the SIRAS protocol the ‘after’ dataset, which was collected at an energy below the absorption peak, was used as native, and the ‘pk’ dataset as the anomalous derivative. In both cases, 100 SHELXD trials in Patterson seeding mode were performed. We used a beta-version of SHELXE (Sheldrick, 2010) to calculate initial phases and improved phases, after density modification was carried out using the sphere of influence method. This newest version of SHELXE includes an autotracing feature (three cycles of autotracing alternating with new phase calculation and density modification) which was used to calculate initial phases and perform 100 cycles of density modification. Initial phases, prior to density modification, were obtained using SHELXE with no density modification cycles (-m0 flag). […]

Pipeline specifications

Software tools XDS, SHELX
Applications Small-angle scattering, Protein structure analysis
Organisms Ruminiclostridium thermocellum
Diseases Tuberculosis
Chemicals Selenium, Selenomethionine