Computational protocol: Ultra-high resolution X-ray structures of two forms of human recombinant insulin at 100 K

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Protocol publication

[…] Manual processing of the data was carried out using XDS [] to integrate and Aimless [] to scale and merge intensities. The purpose of manual scaling was to optimise the included data to maximise the final resolution to 0.92 Å. [...] Molecular replacement was carried out with the published structure 3E7Y as a search model in the program MOLREP [], followed by ten cycles of least squares refinement using the program REFMAC [].Further details can be found in Additional file : Table S1. [...] Model inspection and rebuilding were performed using the program WinCoot 0.7 [] and further isotropic refinement was carried out with the program PHENIX []. Water molecules were added at the end of refinement using the automated method provided in PHENIX. Refinement of the Insugen (I) crystal structure was continued using the program SHELX-97 interfaced with SHELXPRO []. This facilitated the overall inclusion of H atoms and use of anisotropic temperature factors for the non-H atoms. For the protein structure H atoms initially assigned in calculated positions were refined with isotropic thermal parameters. H atoms were not assigned to the waters. During the course of this phase of the analysis several residues were observed in the electron density to have ordered or clear double conformations which were built into the structure and their relative occupancies were included in the refinement summing to 1.0. At the end of the SHELXPRO refinement the R factor and Rfree (all data) were 0.108 and 0.146, respectively. The program MolProbity [] was used for structure validation. Inspection of the Ramachandran plot revealed that 97.53% of the residues are in allowed regions. All coordinates and data have been deposited in the Protein Data Bank, with identification code 5E7W. The final statistics of refinement are summarized in Table . […]

Pipeline specifications

Software tools XDS, CCP4, Molrep, PHENIX, SHELX, MolProbity
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma, Homo sapiens
Diseases Diabetes Mellitus