Fig. 4: Structures of four mtSSU (pre-) translation initiation states.

a Western blot analysis of mitoribosome complexes co-purified via mtIF3^FLAG. FLAG-immunoprecipitation was performed with lysed mitochondria from wild type cells and cells inducibly expressing mtIF3^FLAG. Samples (total = 3%, eluate = 100%) were subsequently analyzed via western blotting with indicated antibodies. Similar results were obtained in n ≥ 3 biologically independent experiments. b Cryo-EM structures of the mtIF3-bound small mitoribosomal subunit (mtSSU) (state D), mtIF2- and mtIF3-bound mtSSU (state E), mtIF2-bound mtSSU (state F), and mtIF2-, mRNA- and fMet-tRNA^Met-bound mtSSU (state G). The 12S rRNA and indicated initiation factors are shown as cartoon and the remaining mitoribosomal proteins (MRPs) are indicated as white transparent surface. 12S rRNA: red, mtIF3: yellow, mtIF2: blue, fMet-tRNA^Met: light yellow, mS37: beige. c Zoomed-in view of the codon-anticodon interaction in state G. Coloring as in (b) with mRNA backbone being depicted in purple and nucleotides being highlighted in red (A), blue (U), green (G), and yellow (C). The 12S rRNA and MRPs are indicated as transparent surface. The trajectory of the mRNA is shown by superimposing with a known IC state (7PO2¹⁹;) and depicted as transparent cartoon. d–g Views of the foot region in each state, showing the densities for MTG3^NTD and for h44 from the 15 Å low-pass filtered maps D1-G1. Mature h44 is depicted by red dashed lines and would clash with MTG^NTD.