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- PDB-4d5n: Cryo-EM structures of ribosomal 80S complexes with termination fa... -
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Basic information
Entry | Database: PDB / ID: 4d5n | |||||||||
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Title | Cryo-EM structures of ribosomal 80S complexes with termination factors and cricket paralysis virus IRES reveal the IRES in the translocated state | |||||||||
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![]() | RIBOSOME/RNA / RIBOSOME-RNA COMPLEX / CRPV IRES / RIBOSOME / TERMINATION / RELEASE FACTORS | |||||||||
Function / homology | ![]() translation termination factor activity / translation release factor complex / cytoplasmic translational termination / translation release factor activity / regulation of translational termination / protein methylation / translation release factor activity, codon specific / sequence-specific mRNA binding / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / aminoacyl-tRNA hydrolase activity ...translation termination factor activity / translation release factor complex / cytoplasmic translational termination / translation release factor activity / regulation of translational termination / protein methylation / translation release factor activity, codon specific / sequence-specific mRNA binding / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / aminoacyl-tRNA hydrolase activity / Protein hydroxylation / Eukaryotic Translation Termination / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / cytosolic ribosome / translational termination / Regulation of expression of SLITs and ROBOs / ribosome binding / RNA binding / cytoplasm / cytosol Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9 Å | |||||||||
![]() | Muhs, M. / Hilal, T. / Mielke, T. / Skabkin, M.A. / Sanbonmatsu, K.Y. / Pestova, T.V. / Spahn, C.M.T. | |||||||||
![]() | ![]() 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 |
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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PDBx/mmCIF format | ![]() | 230 KB | Display | ![]() |
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PDB format | ![]() | 169.8 KB | Display | ![]() |
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-Validation report
Summary document | ![]() | 799.6 KB | Display | ![]() |
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Full document | ![]() | 860.1 KB | Display | |
Data in XML | ![]() | 30.6 KB | Display | |
Data in CIF | ![]() | 44.9 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 2810MC ![]() 2813C ![]() 4d5lC ![]() 4d5yC ![]() 4d61C ![]() 4d67C ![]() 4d66 ![]() 4d68 C: citing same article ( M: map data used to model this data |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 49040.711 Da / Num. of mol.: 1 / Fragment: RESIDUES 5-437 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() |
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#2: RNA chain | Mass: 64363.910 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() |
Sequence details | FIRST CODING TRIPLET MUTATED TO STOP |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: CRICKET PARALYSIS VIRUS IRES RNA BOUND TO MAMMALIAN 80S RIBOSOME AND ERF1 Type: RIBOSOME / Details: MICROGRAPHS SELECTED FOR ASTIGMATISM AND DRIFT |
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Buffer solution | Name: 20 MM TRIS PH 7.5, 100 MM KCL, 1 MM DTT, 2.5 MM MGCL2, 0.5 MM GTP pH: 7.5 Details: 20 MM TRIS PH 7.5, 100 MM KCL, 1 MM DTT, 2.5 MM MGCL2, 0.5 MM GTP |
Specimen | Conc.: 1.38 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Details: HOLEY CARBON |
Vitrification | Instrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Details: LIQUID ETHANE |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Tecnai F20 / Image courtesy: FEI Company |
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Microscopy | Model: FEI TECNAI F20 / Date: Apr 17, 2012 Details: GOOD MICROGRAPHS SELECTED FOR ASTIGMATISM AND DRIFT |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 39000 X / Calibrated magnification: 65520 X / Nominal defocus max: 4000 nm / Nominal defocus min: 2000 nm / Cs: 2 mm |
Specimen holder | Temperature: 77 K |
Image recording | Electron dose: 20 e/Å2 / Film or detector model: KODAK SO-163 FILM |
Image scans | Num. digital images: 366 |
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Processing
EM software |
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CTF correction | Details: DEFOCUS GROUPS | ||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||
3D reconstruction | Method: MULTI-REFERENCE TEMPLATE MATCHING / Resolution: 9 Å / Num. of particles: 109596 / Nominal pixel size: 1.56 Å / Actual pixel size: 1.56 Å Magnification calibration: CROSS- -CORRELATION DENSITIES WITH REFERENCE STRUCTURE Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2810. (DEPOSITION ID: 12907). Symmetry type: POINT | ||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL / Details: METHOD--RIGID BODY, FLEXIBLE FIT | ||||||||||||
Refinement | Highest resolution: 9 Å | ||||||||||||
Refinement step | Cycle: LAST / Highest resolution: 9 Å
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