Extended Data Figure 6. Three dimensional classification of APC/C^{Cdh1.Hsl1.UbcH10-Ub}.

(a) The 3D classification (Cycle 1) started with 477,850 motion corrected particles, which were divided into ten classes. The resultant classes were grouped into five categories: (i) 58.8% in the active ternary state with coactivator and substrate (Hsl1); (ii) 11.9% in the apo inactive state; (iii) 9.4% in a class with Cdh1 bound but with the catalytic module in the apo inactive conformation (hybrid state); (iv) 16.5% with weak Apc2 density and (v) 3.4% were a poor reconstruction due to bad particles. Examination of the hybrid state (iii) showed that density for Apc1^WD40 was absent, explaining the lack of Cdh1-induced conformational change of the catalytic module. Particles in the ternary state (reconstructed to an overall resolution of 4.1 Å) were subjected to further 3D classification (Cycle 2). A major class (class 7, 26.4% of particles) showed improved density for UbcH10 (circled) and Cycle 3 classification was performed on particles in this class. The major class of Cycle 3 (class 5, 25.9% particles) showed further improved UbcH10 density. Further 3D classification of this class did not improve the UbcH10 density. (b) Particles in the best class (Cycle 3, class 5, 19,939 particles) were refined in RELION and resulted in a map at 5.7 Å resolution (Extended Data Fig. 2c). The UbcH10 density was improved by local alignment using a soft mask (indicated by circles) as described in Methods. (c) Enlarged view of UbcH10 density. (d) Enlarged view of the averaged APC/C^{Cdh1.Hsl1.UbcH10-Ub} reconstruction from cycle 1 of the 3D classification (59% of particles). UbcH10 density is circled. (e) Superimposition of classes 6, 7 and 8 of cycle 2 of the 3D classification (from (a)), showing the structural variability of the three 3D classes that indicate UbcH10 density. UbcH10 density is circled.