title: Purification and crystallization of mono-ubiquitylated ubiquitin receptor Rpn10.
abstract: Protein ubiquitylation controls nearly all cellular pathways in eukaryotes. A repertoire of proteins named ubiquitin (Ub) receptors harbouring ubiquitin-binding domains (UBDs) recognize ubiquitylated proteins. These Ub receptors decode the Ub signal by tethering a UBD or UBDs to a functional domain or domains, thus linking the ubiquitylated target to a specific function. The rapid dynamics of ubiquitylation/deubiquitylation has impeded the characterization of ubiquitylated proteins. To bypass this obstacle, a recently developed synthetic system that reconstructs the entire eukaryotic ubiquitylation cascade in Escherichia coli was used to purify the mono-ubiquitylated form of the regulatory proteasomal non-ATPase subunit (Ub-Rpn10) from Saccharomyces cerevisiae. Here, the first crystallization and data collection of Ub-Rpn10 is reported. Purified Ub-Rpn10 was crystallized in 12%(w/v) PEG 20,000, 0.1 M MES pH 6.5 and yielded thin rhombus-shaped crystals. X-ray analysis revealed that these crystals belonged to the monoclinic system C2, with unit-cell parameters a = 107.3, b = 49.7, c = 81.3 Å, α = γ = 90.0, β = 130.5°. A full synchrotron data set has been collected, merged and scaled with a diffraction limit of 3.14 Å.
authors.affiliation: Department of Biochemistry and Molecular Biology and The Institute for Structural Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. talker@post.tau.ac.il
authors.forename: Tal
authors.initials: T
authors.lastname: Keren-Kaplan
authors.forename: Gali
authors.initials: G
authors.lastname: Prag
mesh_terms: D003460:Crystallization; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: C122019:RPN10 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D046988:Proteasome Endopeptidase Complex