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- EMDB-1249: The cryo-EM structure of a translation initiation complex from Es... -

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Basic information

Entry
Database: EMDB / ID: EMD-1249
TitleThe cryo-EM structure of a translation initiation complex from Escherichia coli.
Map dataThis is a difference map, which reveals the non-ribosomal components of the E. coli 70S initiation complex.
Sample
  • Sample: E. coli 70S, IF1, IF3, mRNA, fMet-tRNA, and IF2-GDPNP
  • RNA: fmet-tRNA
  • RNA: mRNA
  • Protein or peptide: beta IF2-GDPNP
  • Protein or peptide: IF1
  • Protein or peptide: IF3
Function / homology
Function and homology information


guanosine tetraphosphate binding / ribosomal small subunit binding / chaperone-mediated protein folding / translation initiation factor activity / response to cold / translational initiation / ribosome binding / rRNA binding / GTPase activity / GTP binding ...guanosine tetraphosphate binding / ribosomal small subunit binding / chaperone-mediated protein folding / translation initiation factor activity / response to cold / translational initiation / ribosome binding / rRNA binding / GTPase activity / GTP binding / membrane / cytosol / cytoplasm
Similarity search - Function
Initiation factor 2 associated domain, bacterial / Bacterial translation initiation factor IF-2 associated region / 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 ...Initiation factor 2 associated domain, bacterial / Bacterial translation initiation factor IF-2 associated region / 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 / : / Elongation factor G domain 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. / Putative DNA-binding domain superfamily / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 / Ribosomal protein S1-like RNA-binding domain / S1 domain / 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 speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 13.8 Å
AuthorsAllen GS / Zavialov A / Gursky R / Ehrenberg M / Frank J
CitationJournal: Cell / Year: 2005
Title: The cryo-EM structure of a translation initiation complex from Escherichia coli.
Authors: Gregory S Allen / Andrey Zavialov / Richard Gursky / Måns Ehrenberg / Joachim Frank /
Abstract: The 70S ribosome and its complement of factors required for initiation of translation in E. coli were purified separately and reassembled in vitro with GDPNP, producing a stable initiation complex ...The 70S ribosome and its complement of factors required for initiation of translation in E. coli were purified separately and reassembled in vitro with GDPNP, producing a stable initiation complex (IC) stalled after 70S assembly. We have obtained a cryo-EM reconstruction of the IC showing IF2*GDPNP at the intersubunit cleft of the 70S ribosome. IF2*GDPNP contacts the 30S and 50S subunits as well as fMet-tRNA(fMet). IF2 here adopts a conformation radically different from that seen in the recent crystal structure of IF2. The C-terminal domain of IF2 binds to the single-stranded portion of fMet-tRNA(fMet), thereby forcing the tRNA into a novel orientation at the P site. The GTP binding domain of IF2 binds to the GTPase-associated center of the 50S subunit in a manner similar to EF-G and EF-Tu. Additionally, we present evidence for the localization of IF1, IF3, one C-terminal domain of L7/L12, and the N-terminal domain of IF2 in the initiation complex.
History
DepositionJul 26, 2006-
Header (metadata) releaseJul 26, 2006-
Map releaseJul 26, 2006-
UpdateMar 19, 2014-
Current statusMar 19, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 8
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-1zo1
  • Surface level: 8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-1zo3
  • Surface level: 8
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-1zo3
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1249.gif

Downloads & links

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Map

FileDownload / File: emd_1249.map.gz / Format: CCP4 / Size: 8.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a difference map, which reveals the non-ribosomal components of the E. coli 70S initiation complex.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.82 Å/pix.
x 130 pix.
= 366.6 Å		2.82 Å/pix.
x 130 pix.
= 366.6 Å		2.82 Å/pix.
x 130 pix.
= 366.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.82 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 7.4 / Movie #1: 8
Minimum - Maximum-55.1922 - 142.330999999999989
Average (Standard dev.)0.138297 (±3.17744)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-65-65-65
Dimensions130130130
Spacing130130130
CellA=B=C: 366.6 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.822.822.82
M x/y/z130130130
origin x/y/z0.0000.0000.000
length x/y/z366.600366.600366.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS-65-65-65
NC/NR/NS130130130
D min/max/mean-55.192142.3310.138

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Supplemental data

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Sample components

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Entire : E. coli 70S, IF1, IF3, mRNA, fMet-tRNA, and IF2-GDPNP

EntireName: E. coli 70S, IF1, IF3, mRNA, fMet-tRNA, and IF2-GDPNP
Components
  • Sample: E. coli 70S, IF1, IF3, mRNA, fMet-tRNA, and IF2-GDPNP
  • RNA: fmet-tRNA
  • RNA: mRNA
  • Protein or peptide: beta IF2-GDPNP
  • Protein or peptide: IF1
  • Protein or peptide: IF3

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Supramolecule #1000: E. coli 70S, IF1, IF3, mRNA, fMet-tRNA, and IF2-GDPNP

SupramoleculeName: E. coli 70S, IF1, IF3, mRNA, fMet-tRNA, and IF2-GDPNP / type: sample / ID: 1000 / Oligomeric state: One of each component binds to the 70S / Number unique components: 7

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Macromolecule #1: fmet-tRNA

MacromoleculeName: fmet-tRNA / type: rna / ID: 1 / Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #5: mRNA

MacromoleculeName: mRNA / type: rna / ID: 5 / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #2: beta IF2-GDPNP

MacromoleculeName: beta IF2-GDPNP / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #3: IF1

MacromoleculeName: IF1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Oligomeric state: 1 / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #4: IF3

MacromoleculeName: IF3 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Oligomeric state: 1 / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: 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

BufferpH: 7.6 / Details: polymix buffer
GridDetails: Quantifoil
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 100 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: 2 second blot

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Electron microscopy

MicroscopeFEI TECNAI F30
TemperatureAverage: 80 K
DateJul 4, 2003
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm / Number real images: 116 / Od range: 1.2 / Bits/pixel: 12
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 39000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.26 mm / Nominal defocus max: 3.93 µm / Nominal defocus min: 0.93 µm / Nominal magnification: 39000
Sample stageSpecimen holder: cryo stage / Specimen holder model: GATAN HELIUM
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

Detailsautomated particle picking followed by manual verification
CTF correctionDetails: defocus groups
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.8 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER
Details: This volume is a difference map constructed by the subtraction of a post-initiation map from the initiation map emd-1248.
Number images used: 20283

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Atomic model buiding 1

SoftwareName: RSRef
DetailsProtocol: real-space refinement of rigid bodies. IF2, P/I site tRNA, and IF1 were initially fit by hand in O then refined by RSRef.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation coefficient
Output model

PDB-1zo1:
IF2, IF1, and tRNA fitted to cryo-EM data OF E. COLI 70S initiation complex

PDB-1zo3:
The P-site and P/E-site tRNA structures fitted to P/I site codon.

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