+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2810 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | Cryo-EM structures of ribosomal 80S complexes with termination factors and cricket paralysis virus IRES reveal the IRES in the translocated state | |||||||||
Map data | Reconstruction of mammalian termination complex with CrPV IRES-RNA and eRF1 | |||||||||
Sample |
| |||||||||
Keywords | CrPV IRES / ribosome / Termination / release factors | |||||||||
Function / homology | Function and homology information translation termination factor activity / cytoplasmic translational termination / translation release factor complex / translation release factor activity / regulation of translational termination / ribosomal subunit / translation release factor activity, codon specific / protein methylation / sequence-specific mRNA binding / aminoacyl-tRNA hydrolase activity ...translation termination factor activity / cytoplasmic translational termination / translation release factor complex / translation release factor activity / regulation of translational termination / ribosomal subunit / translation release factor activity, codon specific / protein methylation / sequence-specific mRNA binding / aminoacyl-tRNA hydrolase activity / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / laminin receptor activity / mammalian oogenesis stage / 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 / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / 90S preribosome / 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 / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / ribosomal small subunit export from nucleus / translation regulator activity / translational termination / laminin binding / 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 / cytosolic ribosome / 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) / rescue of stalled ribosome / 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 / small-subunit processome / protein kinase C binding / positive regulation of apoptotic signaling pathway / positive regulation of protein-containing complex assembly / placenta development / 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 / rhythmic process / positive regulation of canonical Wnt signaling pathway / small ribosomal subunit rRNA binding / ribosome binding / glucose homeostasis / regulation of translation / ribosomal small subunit assembly / virus receptor activity / small ribosomal subunit / T cell differentiation in thymus / cell body / cytosolic small ribosomal subunit / perikaryon / cytoplasmic translation / cytosolic large ribosomal subunit / mitochondrial inner membrane / cell differentiation / postsynaptic density / rRNA binding / ribosome / protein ubiquitination / structural constituent of ribosome / positive regulation of apoptotic process / ribonucleoprotein complex / positive regulation of protein phosphorylation / translation / cell division / DNA repair / mRNA binding / centrosome / ubiquitin protein ligase binding / dendrite / positive regulation of cell population proliferation / synapse / negative regulation of apoptotic process / nucleolus / apoptotic process / protein kinase binding / perinuclear region of cytoplasm / Golgi apparatus / endoplasmic reticulum / DNA binding / RNA binding / zinc ion binding / membrane Similarity search - Function | |||||||||
Biological species | Oryctolagus cuniculus (rabbit) / Homo sapiens (human) / Cricket paralysis virus | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 8.7 Å | |||||||||
Authors | Muhs M / Hilal T / Mielke T / Skabkin MA / Sanbonmatsu KY / Pestova TV / Spahn CMT | |||||||||
Citation | Journal: Mol Cell / Year: 2015 Title: Cryo-EM of ribosomal 80S complexes with termination factors reveals the translocated cricket paralysis virus IRES. Authors: Margarita Muhs / Tarek Hilal / Thorsten Mielke / Maxim A Skabkin / Karissa Y Sanbonmatsu / Tatyana V Pestova / Christian M T Spahn / Abstract: The cricket paralysis virus (CrPV) uses an internal ribosomal entry site (IRES) to hijack the ribosome. In a remarkable RNA-based mechanism involving neither initiation factor nor initiator tRNA, the ...The cricket paralysis virus (CrPV) uses an internal ribosomal entry site (IRES) to hijack the ribosome. In a remarkable RNA-based mechanism involving neither initiation factor nor initiator tRNA, the CrPV IRES jumpstarts translation in the elongation phase from the ribosomal A site. Here, we present cryoelectron microscopy (cryo-EM) maps of 80S⋅CrPV-STOP ⋅ eRF1 ⋅ eRF3 ⋅ GMPPNP and 80S⋅CrPV-STOP ⋅ eRF1 complexes, revealing a previously unseen binding state of the IRES and directly rationalizing that an eEF2-dependent translocation of the IRES is required to allow the first A-site occupation. During this unusual translocation event, the IRES undergoes a pronounced conformational change to a more stretched conformation. At the same time, our structural analysis provides information about the binding modes of eRF1 ⋅ eRF3 ⋅ GMPPNP and eRF1 in a minimal system. It shows that neither eRF3 nor ABCE1 are required for the active conformation of eRF1 at the intersection between eukaryotic termination and recycling. | |||||||||
History |
|
-Structure visualization
Movie |
Movie viewer |
---|---|
Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_2810.map.gz | 86.3 MB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-2810-v30.xml emd-2810.xml | 19.2 KB 19.2 KB | Display Display | EMDB header |
Images | eRF1.png emd_2810.png | 80.3 KB 119.6 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2810 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2810 | HTTPS FTP |
-Validation report
Summary document | emd_2810_validation.pdf.gz | 234.7 KB | Display | EMDB validaton report |
---|---|---|---|---|
Full document | emd_2810_full_validation.pdf.gz | 233.8 KB | Display | |
Data in XML | emd_2810_validation.xml.gz | 6.7 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2810 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2810 | HTTPS FTP |
-Related structure data
Related structure data | 4d5lMC 4d5nMC 4d5yMC 2813C 4d61C 4d67C M: atomic model generated by this map C: citing same article (ref.) |
---|---|
Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|---|
Related items in Molecule of the Month |
-Map
File | Download / File: emd_2810.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Reconstruction of mammalian termination complex with CrPV IRES-RNA and eRF1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.56 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
|
-Supplemental data
-Sample components
-Entire : Ribosomal 80S termination complex with CrPV IRES-RNA and eRF1
Entire | Name: Ribosomal 80S termination complex with CrPV IRES-RNA and eRF1 |
---|---|
Components |
|
-Supramolecule #1000: Ribosomal 80S termination complex with CrPV IRES-RNA and eRF1
Supramolecule | Name: Ribosomal 80S termination complex with CrPV IRES-RNA and eRF1 type: sample / ID: 1000 Oligomeric state: CrPV IRES and eRF1 bound to one 80S ribosome Number unique components: 3 |
---|---|
Molecular weight | Theoretical: 4.5 MDa |
-Supramolecule #1: 80S ribosome
Supramolecule | Name: 80S ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL |
---|---|
Source (natural) | Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit |
Molecular weight | Theoretical: 4.5 MDa |
-Macromolecule #1: eukaryoric release factor 1
Macromolecule | Name: eukaryoric release factor 1 / type: protein_or_peptide / ID: 1 / Name.synonym: eRF1 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes |
---|---|
Source (natural) | Organism: Homo sapiens (human) / synonym: Human |
Molecular weight | Theoretical: 50 KDa |
Recombinant expression | Organism: Escherichia coli BL21 (bacteria) |
Sequence | UniProtKB: Eukaryotic peptide chain release factor subunit 1 |
-Macromolecule #2: Cricket paralysis virus IRES RNA
Macromolecule | Name: Cricket paralysis virus IRES RNA / type: rna / ID: 2 / Name.synonym: CrPV IRES RNA / Classification: OTHER / Structure: OTHER / Synthetic?: No |
---|---|
Source (natural) | Organism: Cricket paralysis virus |
Sequence | String: AAAAAUGUGA UCUUGCUUGU AAAUACAAUU UUGAGAGGUU AAUAAAUUAC AAGUAGUGCU AUUUUUGUAU UUAGGUUAGC UAUUUAGCUU UACGUUCCAG GAUGCCUAGU GGCAGCCCCA CAAUAUCCAG GAAGCCCUCU CUGCGGUUUU UCAGAUUAGG UAGUCGAAAA ...String: AAAAAUGUGA UCUUGCUUGU AAAUACAAUU UUGAGAGGUU AAUAAAUUAC AAGUAGUGCU AUUUUUGUAU UUAGGUUAGC UAUUUAGCUU UACGUUCCAG GAUGCCUAGU GGCAGCCCCA CAAUAUCCAG GAAGCCCUCU CUGCGGUUUU UCAGAUUAGG UAGUCGAAAA ACCUAAGAAA UUUACCUUAA GGCUUCCUCG A |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1.38 mg/mL |
---|---|
Buffer | pH: 7.5 Details: 20 mM Tris pH 7.5, 100 mM KCl, 1 mM DTT, 2.5 mM MgCl2, 0.5 mM GTP |
Grid | Details: Quantifoil grids with additional continuous carbon support |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 85 % / Instrument: FEI VITROBOT MARK II / Method: blot for 2/4 seconds before plunging |
-Electron microscopy
Microscope | FEI TECNAI F20 |
---|---|
Temperature | Min: 77 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 200,000 times magnification |
Details | minimal dose system |
Date | Apr 17, 2012 |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Number real images: 366 / Average electron dose: 20 e/Å2 / Bits/pixel: 16 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 65520 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 39000 |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
Details | The particles were selected using SIGNATURE and processed by using SPIDER and SPARX |
---|---|
CTF correction | Details: Defocus group |
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.7 Å / Resolution method: OTHER / Software - Name: SPIDER, SPARX Details: The particles were selected using SIGNATURE and processed by using SPIDER and SPARX. Number images used: 109596 |
-Atomic model buiding 1
Initial model | PDB ID: 4cxc Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E / Chain - #5 - Chain ID: F / Chain - #6 - Chain ID: G / Chain - #7 - Chain ID: H / Chain - #8 - Chain ID: I / Chain - #9 - Chain ID: J / Chain - #10 - Chain ID: K / Chain - #11 - Chain ID: L / Chain - #12 - Chain ID: M / Chain - #13 - Chain ID: N / Chain - #14 - Chain ID: O / Chain - #15 - Chain ID: P / Chain - #16 - Chain ID: Q / Chain - #17 - Chain ID: R / Chain - #18 - Chain ID: S / Chain - #19 - Chain ID: T / Chain - #20 - Chain ID: U / Chain - #21 - Chain ID: V / Chain - #22 - Chain ID: W / Chain - #23 - Chain ID: X / Chain - #24 - Chain ID: Y / Chain - #25 - Chain ID: Z / Chain - #26 - Chain ID: a / Chain - #27 - Chain ID: b / Chain - #28 - Chain ID: c / Chain - #29 - Chain ID: d / Chain - #30 - Chain ID: e / Chain - #31 - Chain ID: f / Chain - #32 - Chain ID: g / Chain - #33 - Chain ID: 1 |
---|---|
Software | Name: CHIMERA |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
Output model | PDB-4d5l: PDB-4d5n: PDB-4d5y: |
-Atomic model buiding 2
Initial model | PDB ID: 4cxd Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E / Chain - #5 - Chain ID: F / Chain - #6 - Chain ID: G / Chain - #7 - Chain ID: H / Chain - #8 - Chain ID: I / Chain - #9 - Chain ID: J / Chain - #10 - Chain ID: L / Chain - #11 - Chain ID: M / Chain - #12 - Chain ID: N / Chain - #13 - Chain ID: O / Chain - #14 - Chain ID: P / Chain - #15 - Chain ID: Q / Chain - #16 - Chain ID: R / Chain - #17 - Chain ID: S / Chain - #18 - Chain ID: T / Chain - #19 - Chain ID: U / Chain - #20 - Chain ID: V / Chain - #21 - Chain ID: W / Chain - #22 - Chain ID: X / Chain - #23 - Chain ID: Y / Chain - #24 - Chain ID: Z / Chain - #25 - Chain ID: a / Chain - #26 - Chain ID: b / Chain - #27 - Chain ID: c / Chain - #28 - Chain ID: d / Chain - #29 - Chain ID: e / Chain - #30 - Chain ID: f / Chain - #31 - Chain ID: g / Chain - #32 - Chain ID: h / Chain - #33 - Chain ID: i / Chain - #34 - Chain ID: j / Chain - #35 - Chain ID: k / Chain - #36 - Chain ID: l / Chain - #37 - Chain ID: m / Chain - #38 - Chain ID: n / Chain - #39 - Chain ID: o / Chain - #40 - Chain ID: p / Chain - #41 - Chain ID: t / Chain - #42 - Chain ID: u |
---|---|
Software | Name: CHIMERA |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
Output model | PDB-4d5l: PDB-4d5n: PDB-4d5y: |