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- EMDB-2448: Cryo-EM structure of T. thermophilus 30S Translation Initiation c... -

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Basic information

Entry
Database: EMDB / ID: EMD-2448
TitleCryo-EM structure of T. thermophilus 30S Translation Initiation complex
Map dataBacterial 30S Translation Initiation complex from T. thermophilus containing 30S, IF1, IF2, mRNA and fMet-tRNA. The structure have been used for modeling full atom IF2.
Sample
  • Sample: Bacterial 30S Translation Initiation complex from T. thermophilus containing 30S, IF1, IF2, mRNA and fMet-tRNA. The structure have been used for modeling full atom IF2.
  • Complex: 30S
  • Protein or peptide: Translation Initiation Factor 1Initiation factor
  • Protein or peptide: Translation Initiation Factor 2Initiation factor
  • RNA: mRNAMessenger RNA
  • RNA: fMet-tRNA
KeywordsTranslation Initiation complex / 30S / IF2 / IF1 / fMet-tRNA / mRNA / IF2 atomic model
Function / homology
Function and homology information


translation initiation factor activity / ribosome binding / rRNA binding / GTPase activity / GTP binding / cytoplasm
Similarity search - Function
Translation initiation factor IF-1 / Translation initiation factor IF-2, N-terminal / Translation initiation factor IF-2, N-terminal region / Translation initiation factor IF-2, domain II / Initiation factor 2 signature. / Translation initiation factor IF-2, bacterial-like / Translation initiation factor IF- 2, domain 3 / Translation-initiation factor 2 / Translation initiation factor IF- 2 / Translation initiation factor IF-2, domain 3 superfamily ...Translation initiation factor IF-1 / Translation initiation factor IF-2, N-terminal / Translation initiation factor IF-2, N-terminal region / Translation initiation factor IF-2, domain II / Initiation factor 2 signature. / Translation initiation factor IF-2, bacterial-like / Translation initiation factor IF- 2, domain 3 / Translation-initiation factor 2 / Translation initiation factor IF- 2 / Translation initiation factor IF-2, domain 3 superfamily / RNA-binding domain, S1, IF1 type / Translation initiation factor 1A / IF-1 / S1 domain IF1 type profile. / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Small GTP-binding protein domain / Translation protein, beta-barrel domain superfamily / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Translation initiation factor IF-2 / Translation initiation factor IF-1
Similarity search - Component
Biological speciesThermus thermophilus HB8 (bacteria) / synthetic construct (others) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 11.5 Å
AuthorsSimonetti A / Marzi S / Billas IML / Tsai A / Fabbretti A / Myasnikov A / Roblin P / Vaiana AC / Hazemann I / Eiler D ...Simonetti A / Marzi S / Billas IML / Tsai A / Fabbretti A / Myasnikov A / Roblin P / Vaiana AC / Hazemann I / Eiler D / Steitz TA / Puglisi JD / Gualerzi GO / Klaholz BP
CitationJournal: Proc Natl Acad Sci U S A / Year: 2013
Title: Involvement of protein IF2 N domain in ribosomal subunit joining revealed from architecture and function of the full-length initiation factor.
Authors: Angelita Simonetti / Stefano Marzi / Isabelle M L Billas / Albert Tsai / Attilio Fabbretti / Alexander G Myasnikov / Pierre Roblin / Andrea C Vaiana / Isabelle Hazemann / Daniel Eiler / ...Authors: Angelita Simonetti / Stefano Marzi / Isabelle M L Billas / Albert Tsai / Attilio Fabbretti / Alexander G Myasnikov / Pierre Roblin / Andrea C Vaiana / Isabelle Hazemann / Daniel Eiler / Thomas A Steitz / Joseph D Puglisi / Claudio O Gualerzi / Bruno P Klaholz /
Abstract: Translation initiation factor 2 (IF2) promotes 30S initiation complex (IC) formation and 50S subunit joining, which produces the 70S IC. The architecture of full-length IF2, determined by small angle ...Translation initiation factor 2 (IF2) promotes 30S initiation complex (IC) formation and 50S subunit joining, which produces the 70S IC. The architecture of full-length IF2, determined by small angle X-ray diffraction and cryo electron microscopy, reveals a more extended conformation of IF2 in solution and on the ribosome than in the crystal. The N-terminal domain is only partially visible in the 30S IC, but in the 70S IC, it stabilizes interactions between IF2 and the L7/L12 stalk of the 50S, and on its deletion, proper N-formyl-methionyl(fMet)-tRNA(fMet) positioning and efficient transpeptidation are affected. Accordingly, fast kinetics and single-molecule fluorescence data indicate that the N terminus promotes 70S IC formation by stabilizing the productive sampling of the 50S subunit during 30S IC joining. Together, our data highlight the dynamics of IF2-dependent ribosomal subunit joining and the role played by the N terminus of IF2 in this process.
History
DepositionAug 29, 2013-
Header (metadata) releaseSep 4, 2013-
Map releaseJul 9, 2014-
UpdateJul 9, 2014-
Current statusJul 9, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.169
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.169
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j4j
  • Surface level: 0.169
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j4j
  • Surface level: 0.169
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j4j
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2448-30S...

Downloads & links

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Map

FileDownload / File: emd_2448.map.gz / Format: CCP4 / Size: 33.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBacterial 30S Translation Initiation complex from T. thermophilus containing 30S, IF1, IF2, mRNA and fMet-tRNA. The structure have been used for modeling full atom IF2.
Voxel sizeX=Y=Z: 1.82 Å
Density
Contour LevelBy AUTHOR: 0.169 / Movie #1: 0.169
Minimum - Maximum-0.76591331 - 3.60590792
Average (Standard dev.)0.0375132 (±0.22922359)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-103-104-104
Dimensions208208208
Spacing208208208
CellA=B=C: 378.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.821.821.82
M x/y/z208208208
origin x/y/z0.0000.0000.000
length x/y/z378.560378.560378.560
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS-104-103-104
NC/NR/NS208208208
D min/max/mean-0.7663.6060.038

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Supplemental data

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Sample components

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Entire : Bacterial 30S Translation Initiation complex from T. thermophilus...

EntireName: Bacterial 30S Translation Initiation complex from T. thermophilus containing 30S, IF1, IF2, mRNA and fMet-tRNA. The structure have been used for modeling full atom IF2.
Components
  • Sample: Bacterial 30S Translation Initiation complex from T. thermophilus containing 30S, IF1, IF2, mRNA and fMet-tRNA. The structure have been used for modeling full atom IF2.
  • Complex: 30S
  • Protein or peptide: Translation Initiation Factor 1Initiation factor
  • Protein or peptide: Translation Initiation Factor 2Initiation factor
  • RNA: mRNAMessenger RNA
  • RNA: fMet-tRNA

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Supramolecule #1000: Bacterial 30S Translation Initiation complex from T. thermophilus...

SupramoleculeName: Bacterial 30S Translation Initiation complex from T. thermophilus containing 30S, IF1, IF2, mRNA and fMet-tRNA. The structure have been used for modeling full atom IF2.
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
Details: Thermus thermophilus 30S subunit purified from tight couple 70S ribosome
Recombinant expression: No / Ribosome-details: ribosome-prokaryote: SSU 30S, PSR16s
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightExperimental: 850 KDa

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Macromolecule #1: Translation Initiation Factor 1

MacromoleculeName: Translation Initiation Factor 1 / type: protein_or_peptide / ID: 1 / Name.synonym: IF1 / Recombinant expression: Yes
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 8.234 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET
SequenceUniProtKB: Translation initiation factor IF-1

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Macromolecule #2: Translation Initiation Factor 2

MacromoleculeName: Translation Initiation Factor 2 / type: protein_or_peptide / ID: 2 / Name.synonym: IF2 / Recombinant expression: Yes
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 63.178 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET
SequenceUniProtKB: Translation initiation factor IF-2

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Macromolecule #3: mRNA

MacromoleculeName: mRNA / type: rna / ID: 3 / Details: model mRNA / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: synthetic construct (others)
SequenceString:
GGCAAGGAGG UAAAAAUGAA AAAAAAA

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Macromolecule #4: fMet-tRNA

MacromoleculeName: fMet-tRNA / type: rna / ID: 4 / Details: fMet-tRNA charged with Met and formylated / Classification: OTHER / Structure: OTHER / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo 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
GridDetails: QuantiFoil Grid 2/2
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 150 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 82417 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: -3.5 µm / Nominal defocus min: -1.5 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 78 K / Max: 96 K / Average: 88 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DateOct 9, 2011
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Digitization - Sampling interval: 1.82 µm / Number real images: 200 / Average electron dose: 15 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.5 Å / Resolution method: OTHER / Software - Name: EMAN2, IMAGIC / Number images used: 13000
DetailsParticle selection was done semiautomatically with the BOXER routine of the EMAN2 software package followed by visual inspection. Defocus value estimation and contrast transfer function (CTF) correction by phase flipping were done using the program CTFIT from the EMAN2. Sample homogeneity was tested through 3D resampling and classification (3D-SC) using the IMAGIC suite, and structure determination and refinement were done using the EMAN2 software package.

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Atomic model buiding 1

Initial modelPDB ID:

3j4j

SoftwareName: DIREX
Detailsflexible fitting, dynamic elastic network
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: cross-correlation coefficient
Output model

PDB-3j4j:
Model of full-length T. thermophilus Translation Initiation Factor 2 refined against its cryo-EM density from a 30S Initiation Complex map

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