The C85K mutation was inserted in pGEX6p, UbcH5c; the protein was expressed in E. coli with an overnight induction at 16 °C with 0.2 mM IPTG. After lysis in buffer containing 50 mM Hepes (pH 8), 150 mM NaCl, 0.5 mM TCEP the protein was purified using GSH beads (GE Healthcare) and eluted with 50 mM glutathione. The tag was cleaved by GST-3c overnight in dialysis with lysis buffer. After removal of the protease, the uncleaved protein and the GST with GSH beads, the protein was loaded into a S75 column in 20 mM Hepes (pH 8.0), 150 mM NaCl and 1 mM TCEP. The fractions containing the protein were concentrated and stored at −80 °C. The mutant E2 was loaded in a reaction containing 200 μM UbcH5c C85K, 200 μM ubiquitin, 1 μM Uba1 and 50 mM Tris (pH 9.5), 150 mM NaCl, 0.8 mM TCEP, 3 mM ATP and 5 mM MgCl2 (ref. 27 (link)). The reaction was incubated at 35 °C for ~20 h. The E2~Ub complex was purified on a S75 gel-filtration column and the fractions containing only the loaded E2 were concentrated and used for the gel-shift analysis shown in Fig. 4d,e.