A subset of E3s (HECT, RBR) undergo transthiolation where Ubiquitin (Ub) is transferred from the cysteine of the E2 to the catalytic cysteine of the E3. Structural studies indicate that the E2~Ub conjugate is in an open conformation when transthiolation takes place. This contrasts with RING E3s where the closed conformation of the E2~Ub conjugate promotes discharge of Ub to lysine. Using an activity-based probe (ABP) directed against HECT and RBR E3s we identified MYCBP2 as an atypical RING-Cys-Relay (RCR) E3 that undergoes transthiolation with E2 followed by intramolecular Ub relay and Ub transfer to threonine (1, 2). Here I present an X-ray crystal structure of a covalent MYCBP2~E2~Ub (MYCBP2~ABP) complex. This is the first structure of an E3 E2~Ub complex resolving the transient tetrahedral Ub transfer intermediate. The structure shows that the E2~Ub conjugate adopts a closed conformation when it binds to the RING domain of MYCBP2. Altogether, our results show that ABPs can be used to capture native-like E3 E2~Ub complexes and suggest that canonical RING domains can work in tandem with auxiliary domains to promote specific transthiolation. Furthermore, neurodevelopmental defects and delayed injury-induced degeneration in RCR-defective knock-in mice suggest its requirement, and that of substrate esterification activity, for normal neural development and programmed axon degeneration.