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- EMDB-1248: The cryo-EM structure of a translation initiation complex from Es... -

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Basic information

Entry
Database: EMDB / ID: EMD-1248
TitleThe cryo-EM structure of a translation initiation complex from Escherichia coli.
Map data
SampleE. coli 70S, IF1, IF3, mRNA, fMet-tRNA, and IF2-GDPNP:
(nucleic-acidNucleic acid) x 2 / (ribosome-prokaryote) x 2 / beta IF2-GDPNP / IF1 / IF3
Function / homology
Function and homology information


guanosine tetraphosphate binding / ribosomal small subunit binding / translation initiation factor activity / translational initiation / ribosome binding / rRNA binding / GTPase activity / GTP binding / membrane / cytosol
RNA-binding domain, S1, IF1 type / Translation initiation factor aIF-2, bacterial-like / Translation protein, beta-barrel domain superfamily / Translation initiation factor IF-2, N-terminal / Initiation factor 2 associated domain, bacterial / Small GTP-binding protein domain / Translation initiation factor IF-1 / Translational (tr)-type GTP-binding domain / Putative DNA-binding domain superfamily / Nucleic acid-binding, OB-fold ...RNA-binding domain, S1, IF1 type / Translation initiation factor aIF-2, bacterial-like / Translation protein, beta-barrel domain superfamily / Translation initiation factor IF-2, N-terminal / Initiation factor 2 associated domain, bacterial / Small GTP-binding protein domain / Translation initiation factor IF-1 / Translational (tr)-type GTP-binding domain / Putative DNA-binding domain superfamily / Nucleic acid-binding, OB-fold / Translation initiation factor IF- 2 / RNA-binding domain, S1 / Translation initiation factor IF- 2, domain 3 / P-loop containing nucleoside triphosphate hydrolase / Translation initiation factor IF-2, domain 3 superfamily / Translation initiation factor IF-2, domain II
Translation initiation factor IF-2 / Translation initiation factor IF-1
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.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionJun 23, 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: 30
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 30
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-1zo1
  • Surface level: 30
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-1zo1
  • Imaged by Jmol
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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

Downloads & links

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Map

FileDownload / File: emd_1248.map.gz / Format: CCP4 / Size: 8.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
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
Contour LevelPrimary: 63.5 / Movie #1: 30
Minimum - Maximum-66.6267 - 212.468999999999994
Average (Standard dev.)4.02043 (±25.465199999999999)
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-66.627212.4694.020

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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 / Number of components: 7 / Oligomeric State: One of each component binds to the 70S

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Component #1: nucleic-acid, fmet-tRNA

nucleic acidName: fmet-tRNA / Class: T-RNA / Structure: DOUBLE HELIX / Synthetic: No
SourceSpecies: Escherichia coli (E. coli)

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Component #2: ribosome-prokaryote, 30S

Ribosome-prokaryoteName: 30S / Prokaryote: SSU 30S / Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli)

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Component #3: ribosome-prokaryote, 50s

Ribosome-prokaryoteName: 50s / Prokaryote: LSU 50S / Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli)

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Component #4: protein, beta IF2-GDPNP

ProteinName: beta IF2-GDPNP / Oligomeric Details: monomer / Number of Copies: 1 / Recombinant expression: Yes
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #5: protein, IF1

ProteinName: IF1 / Oligomeric Details: 1 / Number of Copies: 1 / Recombinant expression: Yes
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: protein, IF3

ProteinName: IF3 / Oligomeric Details: 1 / Recombinant expression: Yes / Number of Copies: 1
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #7: nucleic-acid, mRNA

nucleic acidName: mRNAMessenger RNA / Class: RNA / Structure: SINGLE STRANDED / Synthetic: Yes
SourceSpecies: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionBuffer solution: polymix buffer / pH: 7.6
Support filmQuantifoil
VitrificationInstrument: FEI VITROBOT / Cryogen name: ETHANE / Temperature: 100 K / Humidity: 90 % / Method: 2 second blot / Details: Vitrification instrument: Vitrobot

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F30 / Date: Jul 4, 2003
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
LensMagnification: 39000 X (nominal), 39000 X (calibrated) / Cs: 2.26 mm / Imaging mode: BRIGHT FIELD / Defocus: 930 - 3930 nm
Specimen HolderHolder: cryo stage / Model: GATAN HELIUM / Temperature: 80
CameraDetector: KODAK SO-163 FILM

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Image acquisition

Image acquisitionNumber of digital images: 116 / Scanner: ZEISS SCAI / Sampling size: 14 µm / Bit depth: 12 / OD range: 1.2

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 20283
Details: automated particle picking followed by manual verification
Applied symmetry: C1 (asymmetric)
3D reconstructionAlgorithm: Reference-based / Software: SPIDER / CTF correction: defocus groups / Resolution: 13.8 Å / Resolution method: FSC 0.5
Details: The falloff of Fourier amplitudes toward higher spatial frequencies was corrected using the x-ray solution scattering intensity distribution of 70S ribosomes from E. coli during each round of refinement.

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Atomic model buiding

Modeling #1Software: RSRef / Refinement protocol: rigid body / Target criteria: correlation coefficient / Refinement space: REAL
Details: Protocol: real-space refinement of rigid bodies. IF2 and IF1 were initially fit by hand in O then refined by RSRef
Output model

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