Fig. 4: Cryo-EM structure of the RNF213-UBE2L3 transthiolation complex.

a Schematic of the covalent RNF213-UBE2L3~Ub complex, mimicking the transient E2-E3 transfer (transthiolation) intermediate. Inset illustrates complex formation by SDS-PAGE, demonstrating >90% labeling of full-length murine RNF213 (1.5 μM) at the highest ABP concentration (68 μM) in the presence of ATPγS (5 mM). Panel representative of n = 3. b Left, cryo-EM density of RNF213-UBE2L3~Ub complex at 3.5 Å resolution. A distinctive density appears between the E3 shell and the CTD ___domain of RNF213, into which UBE2L3 can be unambiguously docked. Right, ribbon representation of the RNF213-UBE2L3 model. Details about the cryo-EM reconstruction are given in Supplementary Fig. 4. c Domain architecture of RNF213. d Close-up view of the E3 module with bound UBE2L3 (color coded according to E3 portions, and UBE2L3 shown in light blue). The two interfaces with CTD and E3-shell are shown enlarged in insets. Comparison with E2 interfaces from other E3 enzymes is given in Supplementary Fig. 5. e Autoubiquitination assay used to validate the structurally determined UBE2L3 binding site. f Autoubiquitination assay with various deletion mutants of RNF213 when partnered with UBE2L3 or UBE2D3. Δ338 assesses the role of the N-terminal residues that were disordered in the cryo-EM structure. g Ubiquitination and lipid ubiquitination assays with RNF213 deletion mutants (auto-ubiquitination, top panel; lipid A ubiquitination, lower panel). Source data are provided as a Source Data file.