Cryo electron microscopy movies of telithromycin bound to the Saccharomyces cerevisiae 80S ribosome (G2400A mutant).
Ontology highlight
ABSTRACT:
SUBMITTER: Timm Oliver Koller
PROVIDER: EMPIAR-11757 | biostudies-other |
REPOSITORIES: biostudies-other
Publications
Context-specific action of macrolide antibiotics on the eukaryotic ribosome.
Svetlov Maxim S MS Koller Timm O TO Meydan Sezen S Shankar Vaishnavi V Klepacki Dorota D Polacek Norbert N Guydosh Nicholas R NR Vázquez-Laslop Nora N Wilson Daniel N DN Mankin Alexander S AS
Nature communications 20210514 1
Macrolide antibiotics bind in the nascent peptide exit tunnel of the bacterial ribosome and prevent polymerization of specific amino acid sequences, selectively inhibiting translation of a subset of proteins. Because preventing translation of individual proteins could be beneficial for the treatment of human diseases, we asked whether macrolides, if bound to the eukaryotic ribosome, would retain their context- and protein-specific action. By introducing a single mutation in rRNA, we rendered yea ...[more]
Similar Datasets
Project description:In an attempt to understand ribosome-induced GTP hydrolysis on eEF2, we determined a 12.6-A cryo-electron microscopy reconstruction of the eEF2-bound 80S ribosome in the presence of aluminum tetrafluoride and GDP, with aluminum tetrafluoride mimicking the gamma-phosphate during hydrolysis. This is the first visualization of a structure representing a transition-state complex on the ribosome. Tight interactions are observed between the factor's G domain and the large ribosomal subunit, as well as between domain IV and an intersubunit bridge. In contrast, some of the domains of eEF2 implicated in small subunit binding display a large degree of flexibility. Furthermore, we find support for a transition-state model conformation of the switch I region in this complex where the reoriented switch I region interacts with a conserved rRNA region of the 40S subunit formed by loops of the 18S RNA helices 8 and 14. This complex is structurally distinct from the eEF2-bound 80S ribosome complexes previously reported, and analysis of this map sheds light on the GTPase-coupled translocation mechanism.
| S-EPMC2990977 | biostudies-literature
Project description:Low-dose images obtained by electron cryo-microscopy (cryo-EM) are often affected by blurring caused by sample motion during electron beam exposure, degrading signal especially at high resolution. We show here that we can align frames of movies, recorded with a direct electron detector during beam exposure of rotavirus double-layered particles, thereby greatly reducing image blurring caused by beam-induced motion and sample stage instabilities. This procedure increases the efficiency of cryo-EM imaging and enhances the resolution obtained in three-dimensional reconstructions of the particle. Using movies in this way is generally applicable to all cryo-EM samples and should also improve the performance of midrange electron microscopes that may have limited mechanical stability and beam coherence.
| S-EPMC3510009 | biostudies-literature
Project description:Phosphofructokinase (Pfk1, EC 2.7.1.11) plays a key regulatory role in the glycolytic pathway. The combination of X-ray crystallographic and biochemical data has provided an understanding of the different conformational changes that occur between the active and inhibited states of the bacterial enzyme, and of the role of the two bacterial effectors. Eukaryotic phosphofructokinases exhibit a far more sophisticated regulatory mechanism, they are more complex structures regulated by a large number of effectors (around 20). Saccharomyces cerevisiae Pfk1 is an 835 kDa hetero-octamer which shows cooperative binding for fructose-6-phosphate (F6P) and non-cooperative binding for ATP. The 3D structure of the F6P-bound state was obtained by cryo-electron microscopy to 1.1 nm resolution. This electron microscopy structure, in combination with molecular replacement using the bacterial enzyme has helped provide initial phases to solve the X-ray structure of the F6P-bound state 12S yeast truncated-tetramer. Biochemical and small-angle X-ray scattering (SAXS) studies had indicated that Pfk1 underwent a large conformational change upon Mg-ATP binding. We have calculated a reconstruction using reference-based 3D projection alignment methods from 0 degrees images acquired from frozen-hydrated preparations of the enzyme in the presence of Mg-ATP. The ATP-bound structure is more extended or open, and the calculated radius of gyration of 7.33 nm (7.0 nm for F6P) is in good agreement with the SAXS data. There is a substantial decrease in the rotational angle between the top and bottom tetramers. Interestingly, all these changes have arisen from a reorientation of the alpha- and beta-subunits in the dimers. The interface region between the alpha- and beta-subunits is now approximately half the size of the one in the F6P-bound structure. This is the first time that the 3D structure of a eukaryotic Pfk1 has been visualized in its T-state (inhibited-state).
| S-EPMC3586225 | biostudies-literature
Project description:Malaria inflicts an enormous burden on global human health. The emergence of parasite resistance to front-line drugs has prompted a renewed focus on the repositioning of clinically approved drugs as potential anti-malarial therapies. Antibiotics that inhibit protein translation are promising candidates for repositioning. We have solved the cryo-EM structure of the cytoplasmic ribosome from the human malaria parasite, Plasmodium falciparum, in complex with emetine at 3.2 Å resolution. Emetine is an anti-protozoan drug used in the treatment of ameobiasis that also displays potent anti-malarial activity. Emetine interacts with the E-site of the ribosomal small subunit and shares a similar binding site with the antibiotic pactamycin, thereby delivering its therapeutic effect by blocking mRNA/tRNA translocation. As the first cryo-EM structure that visualizes an antibiotic bound to any ribosome at atomic resolution, this establishes cryo-EM as a powerful tool for screening and guiding the design of drugs that target parasite translation machinery.
| S-EPMC4086275 | biostudies-literature
Project description:The eukaryotic ribosome consists of a small (40S) and a large (60S) subunit. Rps26 is one of the essential ribosomal proteins of the 40S subunit and is encoded by two almost identical genes, RPS26a and RPS26b. Previous studies demonstrated that Rps26 interacts with the 5' untranslated region of mRNA via the eukaryote-specific 62-YXXPKXYXK-70 (Y62-K70) motif. Those observations suggested that this peptide within Rps26 might play an important and specific role during translation initiation. By using alanine-scanning mutagenesis and engineered strains of the yeast Saccharomyces cerevisiae, we found that single amino acid substitutions within the Y62-K70 motif of Rps26 did not affect the in vivo function of the protein. In contrast, complete deletion of the Y62-K70 segment was lethal. The simultaneous replacement of five conserved residues within the Y62-K70 segment by alanines resulted in growth defects under stress conditions and produced distinct changes in polysome profiles that were indicative of the accumulation of free 60S subunits. Human Rps26 (Rps26-Hs), which displays significant homology with yeast Rps26, supported the growth of an S. cerevisiae Δrps26a Δrps26b strain. However, the Δrps26a Δrps26b double deletion strain expressing Rps26-Hs displayed substantial growth defects and an altered ratio of 40S/60S ribosomal subunits. The combined data strongly suggest that the eukaryote-specific motif within Rps26 does not play a specific role in translation initiation. Rather, the data indicate that Rps26 as a whole is necessary for proper assembly of the 40S subunit and the 80S ribosome in yeast. IMPORTANCE Rps26 is an essential protein of the eukaryotic small ribosomal subunit. Previous experiments demonstrated an interaction between the eukaryote-specific Y62-K70 segment of Rps26 and the 5' untranslated region of mRNA. The data suggested a specific role of the Y62-K70 motif during translation initiation. Here, we report that single-site substitutions within the Y62-K70 peptide did not affect the growth of engineered yeast strains, arguing against its having a critical role during translation initiation via specific interactions with the 5' untranslated region of mRNA molecules. Only the simultaneous replacement of five conserved residues within the Y62-K70 fragment or the replacement of the yeast protein with the human homolog resulted in growth defects and caused significant changes in polysome profiles. The results expand our knowledge of ribosomal protein function and suggest a role of Rps26 during ribosome assembly in yeast.
| S-EPMC4863615 | biostudies-other
Project description:Here we present for the first time a three-dimensional cryo-EM map of the Saccharomyces cerevisiae respiratory supercomplex composed of dimeric complex III flanked on each side by one monomeric complex IV. A precise fit of the existing atomic x-ray structures of complex III from yeast and complex IV from bovine heart into the cryo-EM map resulted in a pseudo-atomic model of the three-dimensional structure for the supercomplex. The distance between cytochrome c binding sites of complexes III and IV is about 6 nm, which supports proposed channeling of cytochrome c between the individual complexes. The opposing surfaces of complexes III and IV differ considerably from those reported for the bovine heart supercomplex as determined by cryo-EM. A closer association between the individual complex domains at the aqueous membrane interface and larger spaces between the membrane-embedded domains where lipid molecules may reside are also demonstrated. The supercomplex contains about 50 molecules of cardiolipin (CL) with a fatty acid composition identical to that of the inner membrane CL pool, consistent with CL-dependent stabilization of the supercomplex.
| S-EPMC3391107 | biostudies-literature
Project description:Ribosomes, the protein factories of living cells, translate genetic information carried by messenger RNAs into proteins, and are thus involved in virtually all aspects of cellular development and maintenance. The few available structures of the eukaryotic ribosome reveal that it is more complex than its prokaryotic counterpart, owing mainly to the presence of eukaryote-specific ribosomal proteins and additional ribosomal RNA insertions, called expansion segments. The structures also differ among species, partly in the size and arrangement of these expansion segments. Such differences are extreme in kinetoplastids, unicellular eukaryotic parasites often infectious to humans. Here we present a high-resolution cryo-electron microscopy structure of the ribosome of Trypanosoma brucei, the parasite that is transmitted by the tsetse fly and that causes African sleeping sickness. The atomic model reveals the unique features of this ribosome, characterized mainly by the presence of unusually large expansion segments and ribosomal-protein extensions leading to the formation of four additional inter-subunit bridges. We also find additional rRNA insertions, including one large rRNA domain that is not found in other eukaryotes. Furthermore, the structure reveals the five cleavage sites of the kinetoplastid large ribosomal subunit (LSU) rRNA chain, which is known to be cleaved uniquely into six pieces, and suggests that the cleavage is important for the maintenance of the T. brucei ribosome in the observed structure. We discuss several possible implications of the large rRNA expansion segments for the translation-regulation process. The structure could serve as a basis for future experiments aimed at understanding the functional importance of these kinetoplastid-specific ribosomal features in protein-translation regulation, an essential step towards finding effective and safe kinetoplastid-specific drugs.
| S-EPMC3659406 | biostudies-literature
Project description:Enterococcus faecalis is a gram-positive organism responsible for serious infections in humans, but as with many bacterial pathogens, resistance has rendered a number of commonly used antibiotics ineffective. Here, we report the cryo-EM structure of the E. faecalis 70S ribosome to a global resolution of 2.8 Å. Structural differences are clustered in peripheral and solvent exposed regions when compared with Escherichia coli, whereas functional centres, including antibiotic binding sites, are similar to other bacterial ribosomes. Comparison of intersubunit conformations among five classes obtained after three-dimensional classification identifies several rotated states. Large ribosomal subunit protein bL31, which forms intersubunit bridges to the small ribosomal subunit, assumes different conformations in the five classes, revealing how contacts to the small subunit are maintained throughout intersubunit rotation. A tRNA observed in one of the five classes is positioned in a chimeric pe/E position in a rotated ribosomal state. The 70S ribosome structure of E. faecalis now extends our knowledge of bacterial ribosome structures and may serve as a basis for the development of novel antibiotic compounds effective against this pathogen.
| S-EPMC7530986 | biostudies-literature
Project description:Archaeal ribosomes have many domain-specific features; however, our understanding of these structures is limited. We present 10 cryo-electron microscopy (cryo-EM) structures of the archaeal ribosome from crenarchaeota Sulfolobus acidocaldarius (Sac) at 2.7-5.7 Å resolution. We observed unstable conformations of H68 and h44 of ribosomal RNA (rRNA) in the subunit structures, which may interfere with subunit association. These subunit structures provided models for 12 rRNA expansion segments and 3 novel r-proteins. Furthermore, the 50S-aRF1 complex structure showed the unique domain orientation of aRF1, possibly explaining P-site transfer RNA (tRNA) release after translation termination. Sac 70S complexes were captured in seven distinct steps of the tRNA translocation reaction, confirming conserved structural features during archaeal ribosome translocation. In aEF2-engaged 70S ribosome complexes, 3D classification of cryo-EM data based on 30S head domain identified two new translocation intermediates with 30S head domain tilted 5-6° enabling its disengagement from the translocated tRNA and its release post-translocation. Additionally, we observed conformational changes to aEF2 during ribosome binding and switching from three different states. Our structural and biochemical data provide new insights into archaeal translation and ribosome translocation.
| S-EPMC10516650 | biostudies-literature
Project description:SecA is an ATP-dependent molecular motor pumping secretory and outer membrane proteins across the cytoplasmic membrane in bacteria. SecA associates with the protein-conducting channel, the heterotrimeric SecYEG complex, in a so-called posttranslational manner. A recent study further showed binding of a monomeric state of SecA to the ribosome. However, the true oligomeric state of SecA remains controversial because SecA can also form functional dimers, and high-resolution crystal structures exist for both the monomer and the dimer. Here we present the cryo-electron microscopy structures of Escherichia coli SecA bound to the ribosome. We show that not only a monomeric SecA binds to the ribosome but also that two copies of SecA can be observed that form an elongated dimer. Two copies of SecA completely surround the tunnel exit, providing a unique environment to the nascent polypeptides emerging from the ribosome. We identified the N-terminal helix of SecA required for a stable association with the ribosome. The structures indicate a possible function of the dimeric form of SecA at the ribosome.
| S-EPMC3945378 | biostudies-literature