Computational protocol: Role of a non-canonical surface of Rad6 in ubiquitin conjugating activity

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Protocol publication

[…] X-ray diffraction data were collected at a wavelength of 1.03 Å at the GM/CA CAT beamline 23-ID-D at the Advanced Photon Source (APS). The diffraction data were recorded with a PILATUS3 detector and processed with HKL3000 (). The Rad6∼Ub crystal belongs to primitive orthorhombic space group P212121 with one Rad6∼Ub conjugate in the asymmetric unit. The crystal structure of Rad6∼Ub was solved by the molecular replacement method using the program, PHASER-MR (), in the PHENIX suite of programs (). The coordinates of yeast Rad6 (Protein Data Bank (PDB) code: 1AYZ) and human ubiquitin (PDB code: 1UBQ) were used as search models. The initial solution was subject to multiple rounds of crystallographic refinement with phenix.refine from the Phenix suite of programs () and rebuilt to fit the electron density with COOT (). The final model of Rad6∼Ub has an R-factor of 21.0% and Rfree of 26.5% for all data between 41.1 and 2.28 Å resolution. The final model has 149 residues in Rad6 and 78 residues in Ubiquitin including the two additional N-terminal residues from the plasmid vector. The crystallographic parameters and final refinement statistics are summarized in Table . Figures were prepared using PyMOL Molecular Graphics System, Version Schrödinger, LLC. […]

Pipeline specifications

Software tools PHENIX, Coot, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Saccharomyces cerevisiae