Purification and crystallization of mono‐ubiquitylated ubiquitin receptor Rpn10

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 C 2, 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.