[English] 日本語
Yorodumi- EMDB-1671: The structure of a rabbit ribosome with an A-P' hybrid state tRNA... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-1671 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | The structure of a rabbit ribosome with an A-P' hybrid state tRNA and EF-2 | |||||||||
Map data | This is a reconstruction of a rabbit reticulocyte ribosome stalled on a pseudoknot in an mRNA with distorted A-P' hybrid state tRNA and EF-2. | |||||||||
Sample |
| |||||||||
Keywords | Ribosome / mRNA / tRNA / stem-loop | |||||||||
Biological species | Oryctolagus cuniculus (rabbit) / Infectious bronchitis virus | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 15.3 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 |
|
-Structure visualization
Movie |
Movie viewer |
---|---|
Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_1671.map.gz | 556.3 KB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-1671-v30.xml emd-1671.xml | 13.4 KB 13.4 KB | Display Display | EMDB header |
Images | EMD-1671.gif | 76.2 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-1671 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-1671 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|---|
Related items in Molecule of the Month |
-Map
File | Download / File: emd_1671.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | This is a reconstruction of a rabbit reticulocyte ribosome stalled on a pseudoknot in an mRNA with distorted A-P' hybrid state tRNA and EF-2. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 3.33 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
|
-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 and EF2 |
---|---|
Components |
|
-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 and EF2 type: sample / ID: 1000 / Oligomeric state: Ribosome, mRNA, tRNA and EF2 / Number unique components: 4 |
---|
-Supramolecule #1: Ribosome
Supramolecule | Name: Ribosome / type: organelle_or_cellular_component / ID: 1 / Name.synonym: 80S ribosome Details: The sample was purified by affinity chromatography using a tag on the mRNA. Number of copies: 1 / Oligomeric state: 40S and 60S / Recombinant expression: No |
---|---|
Source (natural) | Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit / Cell: Reticulocyte |
-Supramolecule #2: Elongation factor 2
Supramolecule | Name: Elongation factor 2 / type: organelle_or_cellular_component / ID: 2 / Name.synonym: EF2 Details: The sample was purified by affinity chromatography using a tag on the mRNA. Number of copies: 1 / Recombinant expression: No |
---|---|
Source (natural) | Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit / Cell: Reticulocyte |
-Macromolecule #1: Infectious bronchitis virus mRNA
Macromolecule | Name: Infectious bronchitis virus mRNA / type: rna / ID: 1 / Name.synonym: mRNA Details: The sample was purified by affinity chromatography. Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes |
---|---|
Source (natural) | Organism: Infectious bronchitis virus |
-Macromolecule #2: tRNA
Macromolecule | Name: tRNA / type: rna / ID: 2 / Name.synonym: tRNA Details: The sample was purified by affinity chromatography using a tag on the mRNA. Classification: TRANSFER / Structure: OTHER / Synthetic?: No |
---|---|
Source (natural) | Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
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 |
---|---|
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 |
---|---|
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 |
---|---|
Final angle assignment | Details: Spider convention |
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.3 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider, WellMAP Details: Maps were calculated using 20215 out of 64413 particles selected by the A-P-state tRNA and EF2 presence. Presence of the tRNA was proved using multivariate statistical analysis focused ...Details: Maps were calculated using 20215 out of 64413 particles selected by the A-P-state tRNA and EF2 presence. 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: 20215 |
-Atomic model buiding 1
Initial model | PDB ID: 2zkq |
---|---|
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: |
---|---|
Software | Name: URO, Refmac, NMFF |
Details | Protocol: Rigid body, Refmac and normal modes fitting. The tRNA crystal structure was regularized 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: 1s1h |
---|---|
Software | Name: URO, Refmac, NMFF |
Details | Protocol: Rigid body, Refmac and normal modes fitting |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
-Atomic model buiding 4
Initial model | PDB ID: 2zkr |
---|---|
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 |