Computational protocol: Structural basis for ligase-specific conjugation of linear ubiquitin chains by HOIP

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Protocol publication

[…] Crystals of HOIPCBR-C diffracted to 2.44 Å. A data set was collected on beamline IO3 (λ=0.9798 Å) at the Diamond Light Source (Oxford, UK) and processed using XDS. The structure was solved by single wavelength anomalous dispersion phasing using the SeMet-derivative of HOIPCBR-C. Heavy atom search, density modification, and initial model building was performed using Phenix AutoSol. Diffraction data for crystals of HOIPCBR-C (WT) and HOIPCBR-C (H889A) in complex with ubiquitin were collected at beamline IO4 (λ=1.282 Å), and IO4-1 (λ=0.9163 Å), respectively. Data were reduced using Xia2 from the CCP4 suite and the structure of the complex was determined by molecular replacement in Phaser using the apo structure and ubiquitin (1UBQ.pdb) as search models. All models were iteratively improved by manual building in Coot and refined using REFMAC5 and Phenix. The stereochemistry of the final models was 05analyzed with Procheck. The model of apo HOIPCBR-C has 94.8% of residues in favored regions, 4.2% in allowed regions and 1% outliers. The final models of WT HOIPCBR-C/ubiquitin and HOIPCBR-C H889A/ubiquitin have 97.6% and 95.5% of residues in the favored regions of Ramachandran plot, respectively. Structural figures were prepared in Pymol. […]

Pipeline specifications

Software tools XDS, PHENIX, CCP4, Coot, REFMAC5, PROCHECK, PyMOL
Applications Small-angle scattering, Protein structure analysis
Diseases Neoplasms
Chemicals Cysteine, Zinc