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Yorodumi- EMDB-1523: Cryo-EM structure of prokaryotic 30S Translation Initiation Complex. -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-1523 | |||||||||
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Title | Cryo-EM structure of prokaryotic 30S Translation Initiation Complex. | |||||||||
Map data | Cryo-EM map of the T.thermophilus 30S Initiation Complex with Initiation Factors IF1 and IF2, the Initiator fMet-tRNA and an mRNA molecule. | |||||||||
Sample |
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Keywords | bacterial translation initiation / protein synthesis / ribosome / electron microscopy / IF1 / IF2 / fMet-tRNA / 30S initiation complex | |||||||||
Function / homology | Translation initiation factor IF-1 / Translation initiation factor IF-2, bacterial-like / translation initiation factor activity Function and homology information | |||||||||
Biological species | Thermus thermophilus HB8 (bacteria) / Escherichia coli (E. coli) | |||||||||
Method | single particle reconstruction / cryo EM / negative staining / Resolution: 8.7 Å | |||||||||
Authors | Simonetti A / Marzi S / Myasnikov AG / Fabbretti A / Yusupov M / Gualerzi CO / Klaholz BP | |||||||||
Citation | Journal: Nature / Year: 2008 Title: Structure of the 30S translation initiation complex. Authors: Angelita Simonetti / Stefano Marzi / Alexander G Myasnikov / Attilio Fabbretti / Marat Yusupov / Claudio O Gualerzi / Bruno P Klaholz / Abstract: Translation initiation, the rate-limiting step of the universal process of protein synthesis, proceeds through sequential, tightly regulated steps. In bacteria, the correct messenger RNA start site ...Translation initiation, the rate-limiting step of the universal process of protein synthesis, proceeds through sequential, tightly regulated steps. In bacteria, the correct messenger RNA start site and the reading frame are selected when, with the help of initiation factors IF1, IF2 and IF3, the initiation codon is decoded in the peptidyl site of the 30S ribosomal subunit by the fMet-tRNA(fMet) anticodon. This yields a 30S initiation complex (30SIC) that is an intermediate in the formation of the 70S initiation complex (70SIC) that occurs on joining of the 50S ribosomal subunit to the 30SIC and release of the initiation factors. The localization of IF2 in the 30SIC has proved to be difficult so far using biochemical approaches, but could now be addressed using cryo-electron microscopy and advanced particle separation techniques on the basis of three-dimensional statistical analysis. Here we report the direct visualization of a 30SIC containing mRNA, fMet-tRNA(fMet) and initiation factors IF1 and GTP-bound IF2. We demonstrate that the fMet-tRNA(fMet) is held in a characteristic and precise position and conformation by two interactions that contribute to the formation of a stable complex: one involves the transfer RNA decoding stem which is buried in the 30S peptidyl site, and the other occurs between the carboxy-terminal domain of IF2 and the tRNA acceptor end. The structure provides insights into the mechanism of 70SIC assembly and rationalizes the rapid activation of GTP hydrolysis triggered on 30SIC-50S joining by showing that the GTP-binding domain of IF2 would directly face the GTPase-activated centre of the 50S subunit. | |||||||||
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_1523.map.gz | 1.2 MB | EMDB map data format | |
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Header (meta data) | emd-1523-v30.xml emd-1523.xml | 13.7 KB 13.7 KB | Display Display | EMDB header |
Images | 1523.gif | 48.6 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-1523 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-1523 | HTTPS FTP |
-Validation report
Summary document | emd_1523_validation.pdf.gz | 202 KB | Display | EMDB validaton report |
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Full document | emd_1523_full_validation.pdf.gz | 201.1 KB | Display | |
Data in XML | emd_1523_validation.xml.gz | 5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1523 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1523 | HTTPS FTP |
-Related structure data
Similar structure data |
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-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_1523.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Cryo-EM map of the T.thermophilus 30S Initiation Complex with Initiation Factors IF1 and IF2, the Initiator fMet-tRNA and an mRNA molecule. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 3 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 : Prokaryotic 30S Translation Initiation Complex. T. thermophilus 3...
Entire | Name: Prokaryotic 30S Translation Initiation Complex. T. thermophilus 30S ribosomal subunit complexed with T. thermophilus IF1, T. thermophilus IF2, E. coli fMet-tRNA and mk27 mRNA. |
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Components |
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-Supramolecule #1000: Prokaryotic 30S Translation Initiation Complex. T. thermophilus 3...
Supramolecule | Name: Prokaryotic 30S Translation Initiation Complex. T. thermophilus 30S ribosomal subunit complexed with T. thermophilus IF1, T. thermophilus IF2, E. coli fMet-tRNA and mk27 mRNA. type: sample / ID: 1000 / Oligomeric state: monomer / Number unique components: 5 |
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Molecular weight | Theoretical: 900 KDa |
-Supramolecule #1: 30S
Supramolecule | Name: 30S / type: complex / ID: 1 / Name.synonym: Small ribosomal subunit Details: Thermus thermophilus 30S subunit purified from tight couple 70S ribosome Recombinant expression: No / Ribosome-details: ribosome-prokaryote: SSU 30S |
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Source (natural) | Organism: Thermus thermophilus HB8 (bacteria) |
Molecular weight | Experimental: 850 KDa |
-Macromolecule #1: Translation Initiation Factor IF1
Macromolecule | Name: Translation Initiation Factor IF1 / type: protein_or_peptide / ID: 1 / Name.synonym: IF1 / Details: Thermus thermophilus IF1 / Number of copies: 1 / Oligomeric state: monomeric / Recombinant expression: Yes |
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Source (natural) | Organism: Thermus thermophilus HB8 (bacteria) |
Molecular weight | Theoretical: 8.234 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET30b (Novagen) |
Sequence | GO: translation initiation factor activity / InterPro: Translation initiation factor IF-1 |
-Macromolecule #2: Translation Initiation Factor IF2
Macromolecule | Name: Translation Initiation Factor IF2 / type: protein_or_peptide / ID: 2 / Name.synonym: IF2 / Details: Thermus thermophilus IF2 / Number of copies: 1 / Oligomeric state: monomeric / Recombinant expression: Yes |
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Source (natural) | Organism: Thermus thermophilus HB8 (bacteria) |
Molecular weight | Theoretical: 63.178 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET30b (Novagen) |
Sequence | GO: translation initiation factor activity InterPro: Translation initiation factor IF-2, bacterial-like |
-Macromolecule #3: fMet-tRNAfMet
Macromolecule | Name: fMet-tRNAfMet / type: rna / ID: 3 / Name.synonym: fMet-tRNA Details: Escherichia coli fMet-tRNAfMet (formylated and aminoacilated in vitro) Classification: TRANSFER / Structure: SINGLE STRANDED / Synthetic?: No |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 25.167 KDa |
Sequence | String: CGCGGGGUGG AGCAGCCUGG UAGCUCGUCG GGCUCAUAAC CCGAAGAUCG UCGGUUCAAA UCCGGCCCCC GCAACCA |
-Macromolecule #4: Messanger RNA
Macromolecule | Name: Messanger RNA / type: rna / ID: 4 / Name.synonym: mRNA / Details: model mRNA / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 8.948 KDa |
Sequence | String: GGCAAGGAGG UAAAAAUGAA AAAAAAA |
-Experimental details
-Structure determination
Method | negative staining, cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.5 mg/mL |
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Buffer | pH: 7.5 Details: 10 mM Hepes (pH 7.5), 70 mM NH4Cl, 30 mM KCl, 8 mM MgAc2 and 1 mM DTT |
Staining | Type: NEGATIVE / Details: no staining, cryo-EM with holey carbon grids |
Grid | Details: 300 mesh Cu/Rh |
Vitrification | Cryogen name: ETHANE / Chamber temperature: 77 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: home-made cryo-plunger / Method: Blot for 2 seconds before plunging |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Temperature | Average: 77 K |
Alignment procedure | Legacy - Astigmatism: lens astigmatism was corrected at 50,000 times magnification |
Date | Jun 22, 2006 |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 5 µm / Number real images: 80 / Average electron dose: 20 e/Å2 / Od range: 1.8 / Bits/pixel: 16 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 51484 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder: Side entry liquid nitrogen-cooled cryo specimen holder Specimen holder model: GATAN LIQUID NITROGEN |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
Details | The particles were selected using a semi-automatic selection program (BOXER from the EMAN package). The selection has been then manually refined. |
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CTF correction | Details: individual particles |
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: BKPR / Details: exact filtered back-projection / Number images used: 32000 |
Final angle assignment | Details: beta gamma |