Computational protocol: Insights into Mucopolysaccharidosis I from the structure and action of α-L-Iduronidase

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Protocol publication

[…] X-ray diffraction data of all native crystals were collected on beam lines 08B1 and 08ID at the Canadian Light Source, and the datasets of three inhibitor-IDUA complexes were collected on beamline 11-1 at the Stanford Synchrotron Radiation Lightsource. The R3 Hg-derivative SAD dataset was collected at the Hg peak energy of 1.0064Å. We collected 1000 frames with an oscillation angle of 1.2° per image and a crystal-to-detector distance of 400 mm to produce a high-redundancy dataset for the SAD phasing. The R3 native dataset was collected with a crystal-to-detector distance of 320 mm and a wavelength of 0.9793Å. We collected 360 frames with an oscillation angle of 1°. The P21 native dataset was collected using a Mar325 detector on beamline 08ID with a crystal-to-detector distance of 300mm and an oscillation angle of 0.5° per frame (covering a total oscillation range of 180°). Three inhibitor-IDUA complexes datasets were collected at a wavelength of 0.97945Å and 180 images were collected with an oscillation angle of 1°.The raw data of R3 native, R3 Hg-SAD, and 5F-IdoAF-IDUA complex were indexed, integrated, and scaled with the XDS suite of programs, and the data of native P21, 2F-IdoAF-IDUA, and IdoA-DNJ-IDUA complexes were processed with HKL-2000. [...] The heavy-atom coordinates, phases and density modification map were calculated by autosol of the Phenix program suite. Four distinct Hg atoms were observed per asymmetric unit. The resultant phasing figure of merit was 0.27. After the density modification, the resulting high-quality electron density map was used for the model-building with ARP/wARP. After ARP/wARP, 90% of the amino-acid residues were in density and the remaining amino acids and the carbohydrate residues were manually built using the program Coot and refined with the program REFMAC5,. The correct side chain orientations of His, Asn, and Gln have been checked by REDUCE. The high resolution P21 native structure was solved by molecular replacement using the program Phaser and the R3 structure solution as the search model. TLS refinement was performed using 12 TLS groups determined by the TLS Motion Determination (TLSMD) server. The stereochemical quality of the protein model was checked using PROCHECK. In the P21 crystal form, 99.4% of the residues were in the favored regions of the Ramachandran plot without any outliers. In the R3 crystal form, 99.4% of the residues were in favored regions of the Ramachandran plot with 0.4% outliers. […]

Pipeline specifications

Software tools XDS, HKL-2000, PHENIX, ARP/wARP, Coot, REFMAC5, PROCHECK
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens, Arabidopsis thaliana
Diseases Mucopolysaccharidosis I, Lysosomal Storage Diseases, Genetic Diseases, Inborn
Chemicals Iduronic Acid