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Yorodumi- EMDB-3040: Cryo-EM structure of a mammalian ribosomal termination complex wi... -
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-Basic information
Entry | Database: EMDB / ID: EMD-3040 | |||||||||
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Title | Cryo-EM structure of a mammalian ribosomal termination complex with ABCE1, eRF1(AAQ) and the UGA stop codon | |||||||||
Map data | Reconstruction of a mammalian 80S ribosome-nascent chain complex containing the UGA stop codon bound to eRF1(AAQ) and ABCE1 | |||||||||
Sample |
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Keywords | ribosome / translation termination / release factor / stop codon / decoding | |||||||||
Function / homology | Function and homology information negative regulation of endoribonuclease activity / CTPase activity / translation termination factor activity / cytoplasmic translational termination / translation release factor complex / translation release factor activity / regulation of translational termination / OAS antiviral response / ribosomal subunit / translation release factor activity, codon specific ...negative regulation of endoribonuclease activity / CTPase activity / translation termination factor activity / cytoplasmic translational termination / translation release factor complex / translation release factor activity / regulation of translational termination / OAS antiviral response / ribosomal subunit / translation release factor activity, codon specific / ribosome disassembly / protein methylation / sequence-specific mRNA binding / aminoacyl-tRNA hydrolase activity / regulation of G1 to G0 transition / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / positive regulation of intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / regulation of translation involved in cellular response to UV / protein-DNA complex disassembly / positive regulation of DNA damage response, signal transduction by p53 class mediator resulting in transcription of p21 class mediator / mammalian oogenesis stage / G1 to G0 transition / activation-induced cell death of T cells / Protein hydroxylation / positive regulation of signal transduction by p53 class mediator / ubiquitin ligase inhibitor activity / Eukaryotic Translation Termination / phagocytic cup / ribosomal small subunit binding / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / 90S preribosome / endoribonuclease inhibitor activity / TOR signaling / endonucleolytic cleavage to generate mature 3'-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / T cell proliferation involved in immune response / erythrocyte development / cellular response to actinomycin D / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / negative regulation of ubiquitin-dependent protein catabolic process / ribosomal subunit export from nucleus / ribosomal small subunit export from nucleus / translation regulator activity / translational termination / rough endoplasmic reticulum / endonucleolytic cleavage in ITS1 to separate SSU-rRNA from 5.8S rRNA and LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / gastrulation / MDM2/MDM4 family protein binding / maturation of LSU-rRNA / cytosolic ribosome / DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest / class I DNA-(apurinic or apyrimidinic site) endonuclease activity / DNA-(apurinic or apyrimidinic site) lyase / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / ribonucleoside triphosphate phosphatase activity / rescue of stalled ribosome / ribosomal large subunit biogenesis / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of SSU-rRNA / cellular response to leukemia inhibitory factor / positive regulation of translation / small-subunit processome / translational initiation / protein kinase C binding / positive regulation of apoptotic signaling pathway / positive regulation of protein-containing complex assembly / placenta development / cellular response to gamma radiation / mRNA 5'-UTR binding / transcription coactivator binding / spindle / Regulation of expression of SLITs and ROBOs / cytoplasmic ribonucleoprotein granule / modification-dependent protein catabolic process / G1/S transition of mitotic cell cycle / rRNA processing / protein tag activity / ribosomal small subunit biogenesis / Interferon alpha/beta signaling / rhythmic process / positive regulation of canonical Wnt signaling pathway / small ribosomal subunit rRNA binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / ribosome biogenesis / ribosome binding / glucose homeostasis / regulation of translation / ribosomal small subunit assembly / ribosomal large subunit assembly / 4 iron, 4 sulfur cluster binding / small ribosomal subunit / T cell differentiation in thymus / large ribosomal subunit rRNA binding / cell body / 5S rRNA binding / cytosolic small ribosomal subunit / perikaryon / cytoplasmic translation / cytosolic large ribosomal subunit / tRNA binding / mitochondrial inner membrane Similarity search - Function | |||||||||
Biological species | Oryctolagus cuniculus (rabbit) / Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.83 Å | |||||||||
Authors | Brown A / Shao S / Murray J / Hegde RS / Ramakrishnan V | |||||||||
Citation | Journal: Nature / Year: 2015 Title: Structural basis for stop codon recognition in eukaryotes. Authors: Alan Brown / Sichen Shao / Jason Murray / Ramanujan S Hegde / V Ramakrishnan / Abstract: Termination of protein synthesis occurs when a translating ribosome encounters one of three universally conserved stop codons: UAA, UAG or UGA. Release factors recognize stop codons in the ribosomal ...Termination of protein synthesis occurs when a translating ribosome encounters one of three universally conserved stop codons: UAA, UAG or UGA. Release factors recognize stop codons in the ribosomal A-site to mediate release of the nascent chain and recycling of the ribosome. Bacteria decode stop codons using two separate release factors with differing specificities for the second and third bases. By contrast, eukaryotes rely on an evolutionarily unrelated omnipotent release factor (eRF1) to recognize all three stop codons. The molecular basis of eRF1 discrimination for stop codons over sense codons is not known. Here we present cryo-electron microscopy (cryo-EM) structures at 3.5-3.8 Å resolution of mammalian ribosomal complexes containing eRF1 interacting with each of the three stop codons in the A-site. Binding of eRF1 flips nucleotide A1825 of 18S ribosomal RNA so that it stacks on the second and third stop codon bases. This configuration pulls the fourth position base into the A-site, where it is stabilized by stacking against G626 of 18S rRNA. Thus, eRF1 exploits two rRNA nucleotides also used during transfer RNA selection to drive messenger RNA compaction. In this compacted mRNA conformation, stop codons are favoured by a hydrogen-bonding network formed between rRNA and essential eRF1 residues that constrains the identity of the bases. These results provide a molecular framework for eukaryotic stop codon recognition and have implications for future studies on the mechanisms of canonical and premature translation termination. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_3040.map.gz | 12.8 MB | EMDB map data format | |
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Header (meta data) | emd-3040-v30.xml emd-3040.xml | 15.2 KB 15.2 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_3040_fsc.xml | 14.4 KB | Display | FSC data file |
Images | emd_3040.tif | 343.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-3040 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-3040 | HTTPS FTP |
-Validation report
Summary document | emd_3040_validation.pdf.gz | 365.7 KB | Display | EMDB validaton report |
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Full document | emd_3040_full_validation.pdf.gz | 365.2 KB | Display | |
Data in XML | emd_3040_validation.xml.gz | 14.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3040 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3040 | HTTPS FTP |
-Related structure data
Related structure data | 3jaiMC 3038C 3039C 3jagC 3jahC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_3040.map.gz / Format: CCP4 / Size: 276 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of a mammalian 80S ribosome-nascent chain complex containing the UGA stop codon bound to eRF1(AAQ) and ABCE1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.34 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Affinity-purified 80S ribosome-nascent chain complex containing t...
Entire | Name: Affinity-purified 80S ribosome-nascent chain complex containing the UGA stop codon bound to eRF1(AAQ) and ABCE1 |
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Components |
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-Supramolecule #1000: Affinity-purified 80S ribosome-nascent chain complex containing t...
Supramolecule | Name: Affinity-purified 80S ribosome-nascent chain complex containing the UGA stop codon bound to eRF1(AAQ) and ABCE1 type: sample / ID: 1000 / Number unique components: 6 |
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Molecular weight | Theoretical: 2.1 MDa |
-Supramolecule #1: 80S ribosome
Supramolecule | Name: 80S ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL |
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Source (natural) | Organism: Oryctolagus cuniculus (rabbit) / synonym: rabbit / Cell: reticulocyte / Location in cell: cytosol |
Molecular weight | Theoretical: 2 MDa |
-Macromolecule #1: eukaryotic release factor 1, G183A, G184A
Macromolecule | Name: eukaryotic release factor 1, G183A, G184A / type: protein_or_peptide / ID: 1 / Name.synonym: eRF1(AAQ) / Number of copies: 1 / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pRSETA |
Sequence | UniProtKB: Eukaryotic peptide chain release factor subunit 1 |
-Macromolecule #2: ATP binding cassette E1
Macromolecule | Name: ATP binding cassette E1 / type: protein_or_peptide / ID: 2 / Name.synonym: ABCE1, Rli1 / Recombinant expression: No |
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Source (natural) | Organism: Oryctolagus cuniculus (rabbit) / synonym: rabbit / Location in cell: cytosol |
Sequence | UniProtKB: ATP-binding cassette sub-family E member 1 |
-Macromolecule #4: Sec61-beta
Macromolecule | Name: Sec61-beta / type: protein_or_peptide / ID: 4 / Details: in vitro translated peptide sequence / Number of copies: 1 / Recombinant expression: No |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human |
-Macromolecule #3: messenger RNA
Macromolecule | Name: messenger RNA / type: rna / ID: 3 / Name.synonym: mRNA Details: in vitro transcribed mRNA sequence containing UAA stop codon Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human |
Sequence | String: UCAAAGUUUG AG |
-Macromolecule #5: transfer RNA
Macromolecule | Name: transfer RNA / type: rna / ID: 5 / Name.synonym: tRNA / Classification: OTHER / Structure: OTHER / Synthetic?: No |
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Source (natural) | Organism: Oryctolagus cuniculus (rabbit) / synonym: rabbit |
Molecular weight | Theoretical: 20 KDa |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.4 / Details: 50 mM Hepes, 100 mM KAc, 5 mM MgAc2, 1 mM DTT |
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Grid | Details: R2/2 400 mesh Cu grids with thin continuous carbon support, glow discharged |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III / Method: 30 sec wait time, blot for 3 sec before plunging |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: FEI |
Details | Automated data acquisition using EPU (FEI) |
Date | Apr 17, 2015 |
Image recording | Category: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 1472 / Average electron dose: 30 e/Å2 Details: Every image is the average of 17 frames recorded by the direct electron detector |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 104478 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.6 µm / Nominal defocus min: 1.7 µm / Nominal magnification: 59000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
-Atomic model buiding 1
Initial model | PDB ID: |
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Software | Name: Chimera, Coot |
Refinement | Space: RECIPROCAL / Protocol: FLEXIBLE FIT |
Output model | PDB-3jai: |
-Atomic model buiding 2
Initial model | PDB ID: |
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Software | Name: Chimera, Coot |
Refinement | Space: RECIPROCAL / Protocol: FLEXIBLE FIT |
Output model | PDB-3jai: |
-Atomic model buiding 3
Initial model | PDB ID: |
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Software | Name: Chimera, Coot |
Refinement | Space: RECIPROCAL / Protocol: FLEXIBLE FIT |
Output model | PDB-3jai: |
-Atomic model buiding 4
Initial model | PDB ID: |
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Software | Name: Chimera, Coot |
Details | Sequence was modified in Coot to agree with rabbit sequence |
Refinement | Space: RECIPROCAL / Protocol: FLEXIBLE FIT |
Output model | PDB-3jai: |
-Atomic model buiding 5
Initial model | PDB ID: |
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Software | Name: Chimera, Coot |
Details | Sequence was modified in Coot to agree with the most prevalent tRNA sequence for each particular codon |
Refinement | Space: RECIPROCAL / Protocol: FLEXIBLE FIT |
Output model | PDB-3jai: |