Gel panels in this figure are representative of two independent experiments; n = 2. a, Our strategy for trapping a mimic of the transient neddylated CRL E2~Ub–substrate complex requires that the E2 UBE2D contain only a single cysteine at the active site. However, UBE2D contains three additional cysteines (Cys21, Cys107 and Cys111). Standard replacements of cysteine by serine or alanine severely compromised activity. On the basis of the structural locations of these cysteines, we presumed that their mutation hindered formation of the RING-activated, closed, active UBE2D~Ub conformation28,29,30. We thus devised a systematic structure- and random-based approach to identify suitable replacements that qualitatively maintain wild-type levels of activity with neddylated CRLs. Structural analysis showed that Cys21 and Cys107 are in close proximity, such that mutation of both residues to alanine may generate a destabilizing cavity at this site. Combining UBE2D2(C107A) with Cys21 mutated to isoleucine, leucine or valine to compensate for the reduced hydrophobic volume led to the identification of C21I(C107A) as a suitable version for testing all other possible replacements for Cys111. A similar approach was taken for UBE2D3. A total of 48 different versions of UBE2D were tested to identify the UBE2D(C21I/C107A/C111D) mutant for chemical trapping at the remaining active site cysteine. b, Top, schematic of pulse-chase assay testing intrinsic activation of thioester-linked UBE2D~Ub intermediates. Although this is often tested by monitoring RING-dependent discharge of ubiquitin from UBE2D to free lysine, RBX1 RING-dependent activity is limited in this assay owing to sequence constraints imposed by the requirements for binding to partners other than UBE2D39. Nonetheless, substrate-independent activation of UBE2D~Ub can be readily visualized using CUL1 complexed with a previously described hyperactive mutant RBX1(N98R)39, and high enzyme and lysine concentrations. UBE2D~Ub generated in a pulse reaction was mixed with NEDD8-modified CUL1–RBX1 (shown here with the N98R mutant) and free lysine, and ubiquitin discharge was monitored over time by Coomassie-stained SDS–PAGE (as shown by the representative gel at the bottom) demonstrating that standard serine or alanine mutations of noncatalytic cysteines compromised activity (shown for the mutant C21A/C107A/C111S), whereas the optimized mutant (C21I/C107A/C111D) retains activity similar to that of the wild type. c, Overview of the generation of our stable proxy for the phosphorylated IκBα substrate intermediate linked at a single atom, and comparison to the previous method used to visualize noncanonical Lys sumoylation63. d, Experiment validating our stable proxy for the UBE2D~Ub-phosphorylated IκBα substrate intermediate linked at a single atom, based on the hypothesis that its simultaneous occupation of the binding sites for the UBE2D~Ub intermediate and substrate should result in more potent inhibition of a neddylated CRL1β-TRCP-dependent substrate priming reaction compared to the individual constituents of the complex. e, Cryo-EM reconstruction of neddylated CRL1β-TRCP2 (with full-length, dimeric β-TRCP2) bound to a mimic of UBE2D2~Ub–IκBα generated by adapting the method used previously to visualize noncanonical lysine sumoylation63. Ubiquitin is isopeptide-bonded to the substituted residue of a UBE2D(L119K) mutant, and a cysteine residue that replaces the acceptor in the substrate is disulfide-bonded to the catalytic cysteine of UBE2D2. This electron microscopy map visualizes the catalytic architecture of dimeric CRL1β-TRCP2 in which the dimerization domain agrees well with the previous crystal structure27, and its linked NEDD8 (circled in yellow) is bound to the backside of UBE2D, but the donor ubiquitin (absent from the region circled in orange) was not visible—presumably owing to inadequacies of the method used to generate this mimic of the catalytic intermediate, in which the ubiquitin and substrate are not both simultaneously linked to the UBE2D catalytic cysteine. Variations between the two protomers of the dimer also exacerbated sample heterogeneity. f, Cryo-EM reconstruction of neddylated CRL1β-TRCP1∆D (with monomeric version of β-TRCP1, from residue 175 to the C terminus20) bound to our newly developed proxy for the UBE2D3~Ub–IκBα intermediate. The phospho-IκBα peptide-substrate-bound β-TRCP–SKP1–CUL1–RBX1–NEDD8–UBE2D portion of this map superimposes with the map for the dimeric complex shown in e, but here the entire complex is visible—including both the NEDD8 (circled in yellow) and donor ubiquitin (circled in orange). g, To further increase cryo-EM sample homogeneity, we considered that the RBX1 RING sequence represents a compromise to meet requirements for its many different catalytic activities achieved with neddylation E2s, various ubiquitin carrying enzymes, and regulators including the inhibitor GLMN72. Therefore, we introduced a second RBX1 linchpin residue via mutation (N98R), which has previously been shown to improve neddylated CRL and UBE2D-dependent substrate priming at the expense of other RBX1-dependent functions (for example, with UBE2M and UBE2R2)39. A Coomassie-stained SDS–PAGE gel from an assay for the intrinsic activity of UBE2D~Ub is shown, showing enhanced neddylated CRL-dependent activation of discharge to free lysine with the RBX1 N98R mutation. h, i, Cryo-EM reconstructions of neddylated CRL1β-TRCP1∆D with RBX1(N98R) bound to our newly developed proxies for the UBE2D3~Ub–IκBα and UBE2D2~Ub–IκBα intermediates, the latter of which was pursued for high-resolution electron microscopy (final reconstruction refined to 3.7 Å resolution, shown on right).