Extended Data Fig. 6: Map quality, model fitting and molecular interactions of 60S-UFM1-E3UFM1 complexes.
From: UFM1 E3 ligase promotes recycling of 60S ribosomal subunits from the ER
Shown are fits of the E3UFM1 complex model (in ribbons) into cryo-EM maps (transparent surface) of the FLAG-UFL1 native pulldown sample (States 1–3; native) and the in vitro reconstituted sample (state 3; in vitro). a, Views highlighting the interactions of the UFL1 C-terminal regions (UFL1-C) with the 60S. The maps are shown unmodified after refinement (upper row) or low-pass filtered at 5 Å (center and bottom rows) to visualize more flexible parts. Bottom row, close-up of the UFL1-C in center row; alternate angle. UFL1, yellow; 60S, grey. b, Views highlighting the interaction network of CDK5RAP3 and DDRGK1 with the UFL1 scaffold. Upper row; central region of the E3 complex (E3) with multiple interactions between UFL1 (yellow) and CDK3RAP5 (blue) near uL13. Lower row; UFL1/DDRGK1(magenta) interface (pHW complementation). In state 1, these parts of the complex are not resolved. c, Views focusing on the DDRGK1 EBH (upper row) and close-up view on the DDRGK1 region near uL24-conjugated UFM1 (lower row; uL24, light pink; UFM1, green). Here, β-augmentation is predicted by AlphaFold formed by UFM1 and the UFIM-containing linker region between the DDRGK1 EBH and the DDRGK1 WH. The cryo-EM maps were low-pass filtered at 5 Å and show experimental evidence for predicted β-augmentation. Note that in state 1, these parts of the complex are not visualized and in states 1 and 2, the DDRGK1 EBH is not positioned. d, Views focusing on the structured PTC loop region (D416-V448) of the UFL1 disordered domain (N391-F479) identified near the peptidyl transferase center (PTC) of the 60S. In the best resolved density map for this region (State 3 from FLAG-UFL1 pulldown), a clear helical density is present that fits the α-helical part of the N-terminus of this region (State 3, upper right). We clearly observe densities for three basic residues (K417, R422 and R423); UFL1 Y443 engages in stacking interactions with 25S rRNA base A4548 (lower right; see also Fig. 3f).
