+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-1672 | |||||||||
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Title | Structure of a rabbit ribosome with an A-P'' hybrid state tRNA | |||||||||
Map data | This is a reconstruction of a rabbit reticulocyte ribosome stalled on a pseudoknot-containing mRNA with a distorted A-P'' hybrid state tRNA. | |||||||||
Sample |
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Keywords | Ribosome / mRNA / tRNA / pseudoknot / hybrid state | |||||||||
Biological species | Oryctolagus cuniculus (rabbit) / Infectious bronchitis virus | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 14.9 Å | |||||||||
Authors | Flanagan IV JF / Namy O / Brierley I / Gilbert RJC | |||||||||
Citation | Journal: Structure / Year: 2010 Title: Direct observation of distinct A/P hybrid-state tRNAs in translocating ribosomes. Authors: John F Flanagan / Olivier Namy / Ian Brierley / Robert J C Gilbert / Abstract: Transfer RNAs (tRNAs) link the genetic code in the form of messenger RNA (mRNA) to protein sequence. Translocation of tRNAs through the ribosome from aminoacyl (A) site to peptidyl (P) site and from ...Transfer RNAs (tRNAs) link the genetic code in the form of messenger RNA (mRNA) to protein sequence. Translocation of tRNAs through the ribosome from aminoacyl (A) site to peptidyl (P) site and from P site to exit site is catalyzed in eukaryotes by the translocase elongation factor 2 (EF-2) and in prokaryotes by its homolog EF-G. During tRNA movement one or more "hybrid" states (A/P) is occupied, but molecular details of them and of the translocation process are limited. Here we show by cryo-electron microscopy that a population of mammalian ribosomes stalled at an mRNA pseudoknot structure contains structurally distorted tRNAs in two different A/P hybrid states. In one (A/P'), the tRNA is in contact with the translocase EF-2, which induces it. In the other (A/P''), the translocase is absent. The existence of these alternative A/P intermediate states has relevance to our understanding of the mechanics and kinetics of translocation. | |||||||||
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_1672.map.gz | 557.5 KB | EMDB map data format | |
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Header (meta data) | emd-1672-v30.xml emd-1672.xml | 11.9 KB 11.9 KB | Display Display | EMDB header |
Images | EMD-1672.gif | 75.5 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-1672 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-1672 | HTTPS FTP |
-Related 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_1672.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | This is a reconstruction of a rabbit reticulocyte ribosome stalled on a pseudoknot-containing mRNA with a distorted A-P'' hybrid state tRNA. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 3.33 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 : Rabbit reticulocyte ribosome stalled on a pseudoknot-containing m...
Entire | Name: Rabbit reticulocyte ribosome stalled on a pseudoknot-containing mRNA with an A-P'' hybrid-state tRNA |
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Components |
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-Supramolecule #1000: Rabbit reticulocyte ribosome stalled on a pseudoknot-containing m...
Supramolecule | Name: Rabbit reticulocyte ribosome stalled on a pseudoknot-containing mRNA with an A-P'' hybrid-state tRNA type: sample / ID: 1000 / Oligomeric state: Ribosome, mRNA, tRNA / Number unique components: 3 |
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-Supramolecule #1: 80S ribosome
Supramolecule | Name: 80S ribosome / type: organelle_or_cellular_component / ID: 1 / Name.synonym: Ribosome Details: The sample was purified by affinity chromatography using a tagged mRNA. Number of copies: 1 / Oligomeric state: 40S and 60S / Recombinant expression: No |
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Source (natural) | Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit / Cell: Reticulocyte |
-Macromolecule #1: tRNA
Macromolecule | Name: tRNA / type: rna / ID: 1 / Name.synonym: tRNA / Classification: TRANSFER / Structure: OTHER / Synthetic?: No |
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Source (natural) | Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit |
-Macromolecule #2: Infectious bronchitis virus mRNA
Macromolecule | Name: Infectious bronchitis virus mRNA / type: rna / ID: 2 / Name.synonym: mRNA / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes |
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Source (natural) | Organism: Infectious bronchitis virus |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.8 Details: 25mM Hepes-KOH pH 7.8, 5mM MgOAc, 1mM cycloheximide, 150mM KOAc |
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Grid | Details: 300 mesh copper grid with lacey carbon film |
Vitrification | Cryogen name: ETHANE / Chamber temperature: 100 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: Homemade plunger / Method: Blot with Whatman number 1 paper |
-Electron microscopy
Microscope | FEI TECNAI F30 |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 5.3 µm / Nominal defocus min: 0.92 µm / Nominal magnification: 38000 |
Sample stage | Specimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN |
Temperature | Average: 115 K |
Alignment procedure | Legacy - Astigmatism: At 200,000 magnification |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 107 / Bits/pixel: 8 |
Experimental equipment | Model: Tecnai F30 / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: Per micrograph |
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Final angle assignment | Details: Spider convention |
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 14.9 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider, WellMAP Details: Maps were calculated using 24494 out of 64413 particles selected by the A-P-state tRNA. Presence of the tRNA was proved using multivariate statistical analysis focused classification of ...Details: Maps were calculated using 24494 out of 64413 particles selected by the A-P-state tRNA. Presence of the tRNA was proved using multivariate statistical analysis focused classification of particles without previous alignment to a tRNA-containing ribosome. Number images used: 24494 |
-Atomic model buiding 1
Initial model | PDB ID: 2zkq |
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Software | Name: URO |
Details | Protocol: Rigid body in segments. Subregions were defined as described in the paper accompanying this deposition. |
Refinement | Space: RECIPROCAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient |
-Atomic model buiding 2
Initial model | PDB ID: |
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Software | Name: URO, Refmac, NMFF |
Details | Protocol: Rigid body, Refmac and normal modes fitting. The tRNA crystal structure was regularisaed in Refmac (CCP4) and then subjected to normal modes-assisted flexible fitting using the program NMFF (Tama et al.) |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
-Atomic model buiding 3
Initial model | PDB ID: 2zkr |
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Software | Name: URO |
Details | Protocol: Rigid body in segments. Subregions were defined as described in the paper accompanying this deposition. |
Refinement | Space: RECIPROCAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient |