Extended Data Fig. 3: ABPs label MYCBP2(cat) C4520 with high selectivity.

a, Recombinant MYCBP2(cat) was profiled with His-tagged ABPs based on the E2s UBE2D2 (ABP5) and UBE2D3 (ABP6). The experiment was repeated twice with similar results. b, Putative active-site cysteines in MYCBP2 were determined by ABP profiling of a panel of cysteine-to-serine mutants. MYCBP2(cat) mutant (3 μM) was incubated with ABP6 (12 μM) at 30 °C for 4 h. ABP-treated samples were resolved by SDS–PAGE and visualized by Coomassie staining and immunoblotting against the hexahistidine reporter tag on the ABP. Mutation of three cysteine residues (C4506, C4520 and C4537) abolished ABP labelling. The asterisk corresponds to inadvertent cleavage of the hexahistidine tag from the ABP due to trace protease contamination of the E3 preparations. The experiment was repeated twice with similar results. c, Using the pLink software, 38 spectral matches corresponding to cysteine-labelling sites in wild-type MYCBP2_cat were identified. Thirty-six of these corresponded to C4520. One of the two remaining matches corresponded to C4440, a predicted Zn-coordinating residue in the RING domain. The other remaining match corresponded to C4600, which did not significantly affect ABP labelling when mutated. The table lists the predicted and found fragment ions for the representative spectrum depicted in Fig. 2b. The spectrum is for a 5⁺ precursor ion (expected m/z = 614.5094; observed m/z = 614.5088). A mass tolerance of 20 p.p.m. was applied for fragment ion assignment. Experiment was carried out once.