Fig. 2. Cryo-EM of rpl4∆63–87 Nog2-associated mutant particles.

a Deletion of the uL4 tunnel domain (aa 63–87) is lethal. Each strain contains GAL-RPL4 that can be turned off by shifting cells from galactose to glucose-medium, and a plasmid that is constitutively expressing an rpl4 mutant allele. The rpl4-1 and rpl4-2 mutantions are alanine scans of residues 63–68 and 69–74, respectively. Serial dilutions (1:10 to 1:10,000) of cultures were pipetted onto selective medium containing either galactose or glucose, incubated at 30 °C, and imaged after 3 days. b Diagram of the internal loop of uL4 (cyan), including the TD (purple) that extends into the NPET to help create the constriction sites (dotted pink line). Indicated arginine residues (orange) were mutated to glutamate. The TD forms the constriction sites in the NPET (pink dotted line). c Close up view of the exit of the NPET in wild-type Nog2 state 1 and rpl4∆63–87 classes R1 and R2. Gray densities are not present in classes R1 and R2 in the spaces where eL39 and the Nog1 CTD should be. Thus, the exposed cartoon model indicates missing components. d View of rpl4∆63–87 classes R1 and R2 from the subunit interface (left) and solvent side (right). Densities are fitted to the atomic model of wild-type Nog2 state 1 (PDB: 3jct). Exposed cartoon models of Arx1 (blue), the Nog1 CTD (magenta), Bud20 (red), Cgr1 (yellow), and Nug1 (cyan) represent missing densities for each respective protein.