Fig 1.
A variety of unrelated RING domains self-assemble into spherical and ring-shaped bodies, and scaffold multiple RING partner proteins on their surface. (A) Single-particle EM micrographs of RING bodies at a nominal magnification of ×80,000, using uranyl acetate counterstain. (Bottom Right) Micrograph is of RING of Mel18, whereas the rest are of PML, Z, and KAP-1/TIF1β and their site I and site II mutants. (B) Single-particle EM micrographs of BARD1 homomeric bodies, BRCA1 homomeric bodies, BARD1:BRCA1 heteromeric bodies, and BARD1:BRCA1 C64G at a nominal magnification of ×80,000, using methylamine tungstenate counterstain. (C and D) EM micrographs of Z and gold-eIF4E (C) and PML and gold-eIF4E (D), counterstained with uranyl acetate. Multiple gold-eIF4E molecules are scaffolded on the surface of both Z bodies and PML bodies, in agreement with gel filtration measurements (Fig. 7A). Note that heterogeneity in the number of molecules scaffolded by RING bodies is likely due to low (femtomolar) protein concentrations required for single-particle EM measurements that are several orders of magnitude below Kd for the respective associations, as well as stochastic heterogeneity on the microscopic level. The median diameter of gold particles is 6 nm.