Computational protocol: X-ray Structure of a Mammalian Stearoyl-CoA Desaturase

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Protocol publication

[…] X-ray diffraction data were collected at beamline 24ID-C (NE-CAT) at the Advanced Photon Source at Argonne National Laboratory. A data set collected from a single crystal at a wavelength of 1.254 Å with a resolution of 2.8 Å was used for phasing (). The phasing data were processed with XDS and scaled with AIMLESS. The presence of zinc as the predominant ion was confirmed via fluorescence emission spectra using an Amptek SDD fluorescence detector, and analysis of the anomalous signal. A second data set with a resolution of 2.6 Å was collected on a second crystal at a wavelength of 0.9795 Å for use in molecular replacement. The higher resolution data were indexed, integrated, and scaled using HKL2000. The crystals belonged to the space group P212121 with unit cell dimensions of a = 77.06 Å, b = 113.77 Å and c = 141.70 Å. [...] SHELXD/SHELXE found 4 anomalous scatterers per asymmetric unit and the resulting density modified map was used to build an all-helical partial model of one protein molecule. The phased translation function from MOLREP was then used to locate a second protein molecule. This partial model and the heavy atom sites were input into PHASER-EP, which was run in MR-SAD phasing mode. Further density modification was carried out with RESOLVE. The structure model was then further built through successive rounds with COOT and refinement with phenix.refine. This model was then used as the input model for molecular replacement using PHASER with the 2.6 Å diffraction data. The model was completed through successive rounds of model building with COOT and refinement with phenix.refine. The crystallographic map was easily interpretable. In the final stages of refinement, TLS groups were determined using TLSMD and protein geometry was validated with MolProbity. Figures were produced with PyMOL (Schrödinger LLC.). Electrostatic surfaces were generated with Chimera. For , coordinates were used from rat cytochrome b5 (PDB ID 1BFX), which is 100% identical to mouse cytochrome b5 over the depicted residue range. […]

Pipeline specifications

Software tools XDS, CCP4, SHELX, Molrep, Coot, PHENIX, MolProbity, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Mus musculus, Dipturus trachyderma, Homo sapiens
Diseases Diabetes Mellitus, Metabolic Diseases
Chemicals Cholesterol Esters, Oxygen, Palmitoyl Coenzyme A, Triglycerides