Mammalian mitochondrial ribosomes (mitoribosomes) synthesize mitochondrially encoded membrane proteins that are critical for mitochondrial function. Here we present the complete atomic structure of the porcine 55S mitoribosome at 3.8 angstrom resolution by cryo-electron microscopy and chemical cross-linking/mass spectrometry. The structure of the 28S subunit in the complex was resolved at 3.6 angstrom resolution by focused alignment, which allowed building of a detailed atomic structure including all of its 15 mitoribosomal-specific proteins. The structure reveals the intersubunit contacts in the 55S mitoribosome, the molecular architecture of the mitoribosomal messenger RNA (mRNA) binding channel and its interaction with transfer RNAs, and provides insight into the highly specialized mechanism of mRNA recruitment to the 28S subunit. Furthermore, the structure contributes to a mechanistic understanding of aminoglycoside ototoxicity.

Many bacteria contain primitive organelles composed entirely of protein. These bacterial microcompartments share a common architecture of an enzymatic core encapsulated in a selectively permeable protein shell; prominent examples include the carboxysome for CO2 fixation and catabolic microcompartments found in many pathogenic microbes. The shell sequesters enzymatic reactions from the cytosol, analogous to the lipid-based membrane of eukaryotic organelles. Despite available structural information for single building blocks, the principles of shell assembly have remained elusive. We present the crystal structure of an intact shell from Haliangium ochraceum, revealing the basic principles of bacterial microcompartment shell construction. Given the conservation among shell proteins of all bacterial microcompartments, these principles apply to functionally diverse organelles and can inform the design and engineering of shells with new functionalities.

Summary: Mitochondrial ribosomes (mitoribosomes) are extensively modified ribosomes of bacterial descent specialized for the synthesis and insertion of membrane proteins that are critical for energy conversion and ATP production inside mitochondria 1 . Mammalian mitoribosomes, which are composed of 39S and 28S subunits 2 , have diverged dramatically from the bacterial ribosomes from which they are derived, rendering them unique compared to bacterial, eukaryotic cytosolic, and fungal mitochondrial ribosomes [3][4][5] . We have previously determined the architecture of the porcine (Sus scrofa) 39S subunit at 4.9 Å resolution 6 , which is highly homologous to the human mitoribosomal large subunit. Here we present the complete atomic structure of the porcine 39S large mitoribosomal subunit determined in the context of a stalled translating mitoribosome at 3.4 Å resolution by cryo-electron microscopy and chemical crosslinking/mass spectrometry. The structure reveals the locations and the detailed folds of 50 mitoribosomal proteins,shows the highly conserved mitoribosomal peptidyl transferase active site in complex with its substrate tRNAs, and defines the path of the nascent chain in mammalian mitoribosomes along their idiosyncratic exit tunnel. Furthermore, we present evidence that a mitochondrial tRNA has become an integral component of the central protuberance of the 39S subunit where it architecturally substitutes for the absence of the 5S rRNA, a ubiquitous component of all cytoplasmic ribosomes.Main Text: Our previous analysis of the porcine 39S mitoribosomal large subunit at 4.9 Å resolution showed the overall fold of the mitoribosomal 16S rRNA as well as the localization of seven mitoribosomal-specific proteins 6 .However, proteins and protein extensions for which no homology models could be generated could not be modeled at this resolution. Additionally, due to the extensive differences between yeast and mammalian mitoribosomes, the recently reported high-resolution structure of the yeast mitoribosomal large subunit 5 is of limited use for understanding of the mammalian-specific aspects of mitoribosomal structure and function 4,7 .Cryo-EM data of porcine 55S mitoribosomes acquired on a movie modeenabled direct electron detector combined with movie frame realignment to compensate for beam-induced specimen motion 8 and maximum-likelihood based image classification and alignment 9 yielded a 3D-reconstruction of the 55S mitoribosome (Extended Data Fig. 1a, b) at 3.6 Å resolution (FSC = 0.143, "gold standard"). However, due to differences in local resolution (Extended Data Fig. 1c), the quality of the density in the 28S subunit part of the cryo-EM map (28S subunit resolution 4.1 Å) was of insufficient quality for reliable model building and refinement. Therefore, we focused the refinement on the 39S subunit, resulting in an improved 3D-reconstruction of the 39S subunit at 3.4 Å resolution (Extended Data Fig. 2), suitable for de-novo modelbuilding, structure refinement, and validation.We were able to build and ref...