UbcH5B~ubiquitin linkage was performed based on published methods21 (link). Briefly, Ube1, UbcH5B(S22R/C85K) and ubiquitin were mixed and buffer exchanged into 50 mM Tris pH 10, 150 mM NaCl using PD-10 desalting columns (GE Healthcare). 10 mM MgCl2, 5 mM ATP and 1 mM TCEP were added and the protein was incubated at 37°C for 16 h. The completeness of the reaction was monitored using SDS-PAGE and covalently linked UbcH5B~ubiquitin was purified from unreacted proteins and Ube1 using a Superdex 75 10/300 GL size exclusion chromatography column (GE Healthcare) equilibrated in protein buffer.