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...

SummaryTelomerase adds telomeric repeats to chromosome ends to balance incomplete replication. Telomerase regulation is implicated in cancer, aging and other human diseases, but progress towards telomerase clinical manipulation is hampered by the lack of structural data. Here we present the cryo-electron microscopy structure of substrate-bound human telomerase holoenzyme at subnanometer resolution, describing two flexibly RNA-tethered lobes: the catalytic core with telomerase reverse transcriptase (TERT) and conserved motifs of telomerase RNA (hTR), and an H/ACA ribonucleoprotein (RNP). In the catalytic core, RNA encircles TERT, adopting a well-ordered tertiary structure with surprisingly limited protein-RNA interactions. The H/ACA RNP lobe comprises two sets of heterotetrameric H/ACA proteins and one Cajal body protein, TCAB1, representing a pioneering structure of a large eukaryotic family of ribosome and spliceosome biogenesis factors. Our findings provide a structural framework for understanding human telomerase disease mutations and represent an important step towards telomerase-related clinical therapeutics.

Abstract:Mitochondrial ribosomes synthesize a number of highly hydrophobic proteins encoded on the genome of mitochondria, the organelles in eukaryotic cells that are responsible for energy conversion by oxidative phosphorylation. The ribosomes in mammalian mitochondria have undergone massive structural changes throughout their evolution, including rRNA shortening and acquisition of mitochondrial-specific ribosomal proteins. Here, we present the three-dimensional structure of the 39S large subunit of the porcine mitochondrial ribosome determined by cryo-electron microscopy at 4.9 Å resolution. The structure, combined with data from chemical crosslinking and mass spectrometry experiments, reveals the unique features of the 39S subunit at near atomic resolution and provides detailed insight into the architecture of the polypeptide exit site. This region of the mitochondrial ribosome has been dramatically remodeled, providing a specialized platform for the synthesis and membrane insertion of the highly hydrophobic protein components of the respiratory chain. 3Main Text: Mitochondrial ribosomes (mitoribosomes) are responsible for protein synthesis in mitochondria. These organelles of endosymbiotic origin 1 are required for energy conversion by aerobic respiration in eukaryotic cells.Mitoribosomes are more closely related to bacterial ribosomes than to eukaryotic cytosolic ribosomes 2 . However, the mammalian mitoribosome has been strongly altered by acquisition of mitochondrial-specific ribosomal proteins and protein extensions 2-5 , as well as the shortening of the mitochondrial ribosomal RNA (rRNA) 6 . The large 39S subunit of the mammalian mitoribosome catalyzes peptide bond formation during protein synthesis and harbors the nascent polypeptide exit tunnel. The structural evolution of the mammalian mitoribosome was accompanied by a strong functional specialization towards the synthesis of the highly hydrophobic mitochondrial inner membrane proteins 7 . The region around the polypeptide tunnel exit of the mitoribosome serves as a specialized platform for membrane insertion and assembly of these critical mitochondrially-encoded respiratory chain components [7][8][9][10][11] . Defects of the mitochondrial translation system are causally involved in a range of human diseases 12 .While cryo-electron microscopy (cryo-EM) reconstructions of the bovine mitoribosome at 13.5 Å 13 and 12.1 Å 14 resolution have visualized large structural differences compared to the bacterial ribosome, detailed molecular insight into the architecture and arrangement of unique protein and rRNA elements of the mammalian mitoribosome is currently lacking. We have used cryo-EM combined with chemical crosslinking followed by mass spectrometry (CX-MS) to determine the structure of the large subunit of the mammalian 4 mitoribosome, providing insight into its overall structure and into the molecular architecture of the polypeptide exit site in particular. Structure determinationTo obtain structural insights into the mammalian mitochondrial ribosome...

The general transcription factor IID (TFIID) is a critical component of the eukaryotic transcription preinitiation complex (PIC) and is responsible for recognizing the core promoter DNA and initiating PIC assembly. We used cryo-electron microscopy (cryo-EM), chemical crosslinking-mass spectrometry (CX-MS) and biochemical reconstitution to determine the complete molecular architecture of TFIID and define the conformational landscape of TFIID in the process of TATA-box binding protein (TBP) loading onto promoter DNA. Our structural analysis revealed five structural states of TFIID in the presence of TFIIA and promoter DNA, showing that the initial binding of TFIID to the downstream promoter positions the upstream DNA and facilitates scanning of TBP for a TATA-box and the subsequent engagement of the promoter. Our findings provide a mechanistic model for the specific loading of TBP by TFIID onto the promoter.

Eukaryotic ribosome biogenesis depends on several hundred assembly factors to produce functional 40S and 60S ribosomal subunits. The final phase of 60S subunit biogenesis is cytoplasmic maturation, which includes the proofreading of functional centers of the 60S subunit and the release of several ribosome biogenesis factors. We report the cryo-electron microscopy (cryo-EM) structure of the yeast 60S subunit in complex with the biogenesis factors Rei1, Arx1, and Alb1 at 3.4 Å resolution. In addition to the network of interactions formed by Alb1, the structure reveals a mechanism for ensuring the integrity of the ribosomal polypeptide exit tunnel. Arx1 probes the entire set of inner-ring proteins surrounding the tunnel exit, and the C terminus of Rei1 is deeply inserted into the ribosomal tunnel, where it forms specific contacts along almost its entire length. We provide genetic and biochemical evidence that failure to insert the C terminus of Rei1 precludes subsequent steps of 60S maturation.

Mitochondrial ribosomes (mitoribosomes) perform protein synthesis inside mitochondria, the organelles responsible for energy conversion and adenosine triphosphate production in eukaryotic cells. Throughout evolution, mitoribosomes have become functionally specialized for synthesizing mitochondrial membrane proteins, and this has been accompanied by large changes to their structure and composition. We review recent high-resolution structural data that have provided unprecedented insight into the structure and function of mitoribosomes in mammals and fungi.