Fig. 3: Conformational changes for E1–Ub(T)–E2 complexes and coupling of Ub(A) adenylation and Ub(T) transitions to E2 for doubly loaded E1–Ub(T)–E2.
From: Structural basis for transthiolation intermediates in the ubiquitin pathway
a, Superposed models of clusters 1 and 5 for singly loaded E1 with Ub(T) in solid as isosurface rendering of 5 Å lowpass-filtered EM densities showing translation of E1 FCCH and SCCH domains and E2 as indicated by labels and arrows; rotational changes are not labeled. b, Superposed models of cluster 1 and 5 for doubly loaded E1 rendered as in a, highlighting translation of the E1 SCCH domain, E2 and Ub(T). c,d, Electron microscopy densities and models for the adenylation (A) active site (c) and Ub(T) conformations (%) per E1 cluster (d) (Extended Data Fig. 6) indicate that E1 and E2 rotations do not correlate with changes in Ub(T) positioning. e,f, Electron microscopy densities for the adenylation (A) active site (e) and Ub(T) conformations (%) per E1 cluster (f) (Extended Data Fig. 7) indicate that E1 and E2 rotations correlate with loss of PPi in the adenylation site and movement of Ub(T) from donor to acceptor positions. Isosurface levels contoured at 0.23–0.24 (a,b); 1.06, 1.01 and 0.9 for clusters 1, 3 and 5 (c); and 1.19, 1.2 and 1.15 for clusters 1, 3 and 5 (e). Ub(T) conformations (%) per E1 cluster are plotted as a bar graph (Extended Data Fig. 7). g, Stimulation of E1-to-E2 transthiolation by E1 adenylation site ligands. SDS–PAGE gels indicating E1~Ub(T) and E2~Ub(T) over time after initiating reactions with E2. h, Quantification of transthiolation rates from g (Extended Data Fig. 5k and Supplementary Fig. 3). RFU, relative fluorescence units. Green triangle indicates fluorescein. Bars represent mean ± s.d of n = 3 replicates. Statistical differences by two-sided one-way ANOVA with Tukey’s test, ***P < 0.001, **P < 0.01.
