Fig. 4: E2–Ub(T)-E3HECT structures reveal active site remodelling and conformational changes for E2 and Ub(T).
From: Structural basis for transthiolation intermediates in the ubiquitin pathway
a, Schematic of Ub (Ub(T)), E2 (Ubc4) and E3HECT (Pub2) with domains colour-coded and the crosslink indicated by lines between Ub(T), E2 and E3. b, Seven reconstructions obtained for E2–Ub(T)–E3HECT showing conformations of Ub(T) in states 1–7 from donor to intermediate positions (unsharpened maps). c, Reconstructions and models of state 1 and state 7 next to orthogonal views (sharpened maps), highlighting movement of Ub(T). d, Superposed models showing movement of Ub(T) between states 1 and 7. e, Magnified views of transthiolation sites (T-site) with electron microscopy densities for states 1 and 7, showing the cyanomethyldithioacetal mimic of the E2–Ub(T)–E3HECT tetrahedral intermediate and Ub(T) Arg74 side chain. f, Magnified views of areas of interest highlighting differences in E2 contacts with Ub(T) between states 1 and 7 with residues labelled. g, Results of transthiolation assays with E2, Ub and E3 mutations to probe putative protein–protein interactions during transthiolation, quantified after normalization to wild type (WT) with indicated mutations labelled and colour-coded. Densities and models colour-coded according to a. Isosurface levels contoured at 0.47–0.52 (b); at 0.72, 0.75 and 0.71 for state 1 and 0.5, 0.6 and 0.59 for state 7 (c,e,f). Bars represent mean ± s.d of n = 3 replicates (n = 9 and n = 6 for wild type in E2 and E3 mutations sets, respectively). Statistical differences between wild type and mutants by two-tailed unpaired t-test, ***P < 0.001 (Supplementary Fig. 4).
