Journal: 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
Deposition
Jul 26, 2006
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Header (metadata) release
Jul 26, 2006
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Map release
Jul 26, 2006
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Update
Mar 19, 2014
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Current status
Mar 19, 2014
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Entire : E. coli 70S, IF1, IF3, mRNA, fMet-tRNA, and IF2-GDPNP
Entire
Name: 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
Supramolecule
Name: 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
Macromolecule
Name: 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
Macromolecule
Name: mRNA / type: rna / ID: 5 / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
Cryogen 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
Microscope
FEI TECNAI F30
Temperature
Average: 80 K
Date
Jul 4, 2003
Image recording
Category: 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 beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
automated particle picking followed by manual verification
CTF correction
Details: defocus groups
Final reconstruction
Applied 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
Software
Name: RSRef
Details
Protocol: real-space refinement of rigid bodies. IF2, P/I site tRNA, and IF1 were initially fit by hand in O then refined by RSRef.
Refinement
Space: 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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