Extended Data Fig. 2: Superposition of apo-MYCBP2 (PDB 5O6C), E2~Ub bound-MYCBP2 and protein crystal stability.

a, Apo-MYCBP2 residues Asn4379-His4638 were aligned with bound-MYCBP2 residues Asp4387-Asn4636. The RCR, E2 and Ub are in cartoon representation: Coloring (apo-MYCBP2/bound-MYCBP2) zinc ions (light gray/gray), RING domain (light blue/blue), linker helix (pink/purple), helix-turn-helix (light green/green) and tandem cysteine domain (yellow/orange). In the apo structure, 8 residues from the mediator loop are disordered and are represented by a black dashed line. The E2 is colored mauve and Ub is gray. MYCBP2 Residues Ala4518, Gly4527, Cys4520 and Cys4572 are in ball and stick representation. In the bound-MYCBP2 structure E2 residue C85 and the engineered crosslinker are in ball and stick representation. b, Closeup of TC domains, in the E2~Ub bound structure the eight mediator loop residues, that were disordered in the apo structure, adopt a helical conformation in the E2~Ub:RCR transfer complex. c, Closeup of RING domains, in the E2~Ub bound structure the RING domain has twisted towards the linker-helix this results in a 3.0 and 4.1 Å shift of Zn²⁺ 1 and Zn²⁺ 2, respectively. d, Representative view of the E2~Ub-MYCBP2 transfer intermediate. e, Representative view of the E2~Ub-MYCBP2 transfer intermediate colored by B-factors (blue thin-cartoon lowest B-factors to red thick-cartoon highest B-factors) indicates that ubiquitin and the mediator loop are the most disordered components of the complex. f, SDS-PAGE gel of purified ABP-MYCBP2 complex and ABP-MYCBP2 complex recovered from a crystal drop containing the productive conditions (0.85 M sodium citrate, 100 mM sodium chloride, 100 mM Tris-HCl pH 8.0). The ABP-labelled transfer complex is stable during crystallization. Experiment was repeated twice with similar results.