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- EMDB-1523: Cryo-EM structure of prokaryotic 30S Translation Initiation Complex. -

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Basic information

Entry
Database: EMDB / ID: EMD-1523
TitleCryo-EM structure of prokaryotic 30S Translation Initiation Complex.
Map dataCryo-EM map of the T.thermophilus 30S Initiation Complex with Initiation Factors IF1 and IF2, the Initiator fMet-tRNA and an mRNA molecule.
Sample
  • Sample: 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.
  • Complex: 30S
  • Protein or peptide: Translation Initiation Factor IF1
  • Protein or peptide: Translation Initiation Factor IF2
  • RNA: fMet-tRNAfMet
  • RNA: Messanger RNA
Keywordsbacterial translation initiation / protein synthesis / ribosome / electron microscopy / IF1 / IF2 / fMet-tRNA / 30S initiation complex
Function / homologyTranslation initiation factor IF-1 / Translation initiation factor IF-2, bacterial-like / translation initiation factor activity
Function and homology information
Biological speciesThermus thermophilus HB8 (bacteria) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 8.7 Å
AuthorsSimonetti A / Marzi S / Myasnikov AG / Fabbretti A / Yusupov M / Gualerzi CO / Klaholz BP
CitationJournal: 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
DepositionJun 9, 2008-
Header (metadata) releaseJun 9, 2008-
Map releaseApr 1, 2009-
UpdateDec 11, 2013-
Current statusDec 11, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.232454365
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.232454365
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1523.gif

Downloads & links

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Map

FileDownload / File: emd_1523.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of the T.thermophilus 30S Initiation Complex with Initiation Factors IF1 and IF2, the Initiator fMet-tRNA and an mRNA molecule.
Voxel sizeX=Y=Z: 3 Å
Density
Contour Level1: 0.23 / Movie #1: 0.2324544
Minimum - Maximum-3.71055 - 6.47572
Average (Standard dev.)0.0469111 (±0.39919)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-60-60-60
Dimensions120120120
Spacing120120120
CellA=B=C: 360 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z333
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z360.000360.000360.000
α/β/γ90.00090.00090.000
start NX/NY/NZ494949
NX/NY/NZ969696
MAP C/R/S123
start NC/NR/NS-60-60-60
NC/NR/NS120120120
D min/max/mean-3.7116.4760.047

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Supplemental data

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Sample components

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Entire : Prokaryotic 30S Translation Initiation Complex. T. thermophilus 3...

EntireName: 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.
Components
  • Sample: 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.
  • Complex: 30S
  • Protein or peptide: Translation Initiation Factor IF1
  • Protein or peptide: Translation Initiation Factor IF2
  • RNA: fMet-tRNAfMet
  • RNA: Messanger RNA

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Supramolecule #1000: Prokaryotic 30S Translation Initiation Complex. T. thermophilus 3...

SupramoleculeName: 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
Molecular weightTheoretical: 900 KDa

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Supramolecule #1: 30S

SupramoleculeName: 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
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightExperimental: 850 KDa

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Macromolecule #1: Translation Initiation Factor IF1

MacromoleculeName: 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
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 8.234 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET30b (Novagen)
SequenceGO: translation initiation factor activity / InterPro: Translation initiation factor IF-1

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Macromolecule #2: Translation Initiation Factor IF2

MacromoleculeName: 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
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 63.178 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET30b (Novagen)
SequenceGO: translation initiation factor activity
InterPro: Translation initiation factor IF-2, bacterial-like

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Macromolecule #3: fMet-tRNAfMet

MacromoleculeName: 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
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 25.167 KDa
SequenceString:
CGCGGGGUGG AGCAGCCUGG UAGCUCGUCG GGCUCAUAAC CCGAAGAUCG UCGGUUCAAA UCCGGCCCCC GCAACCA

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Macromolecule #4: Messanger RNA

MacromoleculeName: Messanger RNA / type: rna / ID: 4 / Name.synonym: mRNA / Details: model mRNA / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 8.948 KDa
SequenceString:
GGCAAGGAGG UAAAAAUGAA AAAAAAA

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.5
Details: 10 mM Hepes (pH 7.5), 70 mM NH4Cl, 30 mM KCl, 8 mM MgAc2 and 1 mM DTT
StainingType: NEGATIVE / Details: no staining, cryo-EM with holey carbon grids
GridDetails: 300 mesh Cu/Rh
VitrificationCryogen name: ETHANE / Chamber temperature: 77 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: home-made cryo-plunger / Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 51484 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder
Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 77 K
Alignment procedureLegacy - Astigmatism: lens astigmatism was corrected at 50,000 times magnification
DateJun 22, 2006
Image recordingCategory: 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
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: individual particles
Final angle assignmentDetails: beta gamma
Final reconstructionApplied 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
DetailsThe 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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