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- EMDB-1770: 30S-002mRNA (after classification) -

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Basic information

Entry
Database: EMDB / ID: EMD-1770
Title30S-002mRNA (after classification)
Map data30S-002mRNA (after classification)
Sample
  • Sample: 30S.mRNA complex
  • Complex: Ribosome
  • RNA: mRNA
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 21.0 Å
AuthorsJulian P / Milon P / Agirrezabala X / Lasso G / Gil D / Rodnina MV / Valle M
CitationJournal: PLoS Biol / Year: 2011
Title: The Cryo-EM structure of a complete 30S translation initiation complex from Escherichia coli.
Authors: Patricia Julián / Pohl Milon / Xabier Agirrezabala / Gorka Lasso / David Gil / Marina V Rodnina / Mikel Valle /
Abstract: Formation of the 30S initiation complex (30S IC) is an important checkpoint in regulation of gene expression. The selection of mRNA, correct start codon, and the initiator fMet-tRNA(fMet) requires ...Formation of the 30S initiation complex (30S IC) is an important checkpoint in regulation of gene expression. The selection of mRNA, correct start codon, and the initiator fMet-tRNA(fMet) requires the presence of three initiation factors (IF1, IF2, IF3) of which IF3 and IF1 control the fidelity of the process, while IF2 recruits fMet-tRNA(fMet). Here we present a cryo-EM reconstruction of the complete 30S IC, containing mRNA, fMet-tRNA(fMet), IF1, IF2, and IF3. In the 30S IC, IF2 contacts IF1, the 30S subunit shoulder, and the CCA end of fMet-tRNA(fMet), which occupies a novel P/I position (P/I1). The N-terminal domain of IF3 contacts the tRNA, whereas the C-terminal domain is bound to the platform of the 30S subunit. Binding of initiation factors and fMet-tRNA(fMet) induces a rotation of the head relative to the body of the 30S subunit, which is likely to prevail through 50S subunit joining until GTP hydrolysis and dissociation of IF2 take place. The structure provides insights into the mechanism of mRNA selection during translation initiation.
History
DepositionAug 3, 2010-
Header (metadata) releaseSep 24, 2010-
Map releaseAug 12, 2011-
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: 0.34
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.34
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-1770.tif

Downloads & links

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Map

FileDownload / File: emd_1770.map.gz / Format: CCP4 / Size: 8.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation30S-002mRNA (after classification)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.8 Å/pix.
x 130 pix.
= 364. Å		2.8 Å/pix.
x 130 pix.
= 364. Å		2.8 Å/pix.
x 130 pix.
= 364. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.34 / Movie #1: 0.34
Minimum - Maximum-0.298276 - 1.59615
Average (Standard dev.)0.0408365 (±0.143706)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-65-65-65
Dimensions130130130
Spacing130130130
CellA=B=C: 364 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z130130130
origin x/y/z0.0000.0000.000
length x/y/z364.000364.000364.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS-65-65-65
NC/NR/NS130130130
D min/max/mean-0.2981.5960.041

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Supplemental data

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Sample components

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Entire : 30S.mRNA complex

EntireName: 30S.mRNA complex
Components
  • Sample: 30S.mRNA complex
  • Complex: Ribosome
  • RNA: mRNA

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Supramolecule #1000: 30S.mRNA complex

SupramoleculeName: 30S.mRNA complex / type: sample / ID: 1000 / Number unique components: 2

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Supramolecule #1: Ribosome

SupramoleculeName: Ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: SSU 30S
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: mRNA

MacromoleculeName: mRNA / type: rna / ID: 1 / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli)

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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

BufferDetails: 50 mM Tris-HCl, pH 7.5, 70 mM NH4Cl, 30 mM KCl, and 7 mM MgCl2
StainingType: NEGATIVE / Details: Cryo_EM
GridDetails: Quantifoil grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: OTHER / Details: Vitrification instrument: Vitrobot

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Electron microscopy

MicroscopeJEOL 2200FS
Image recordingDigitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 50000
Sample stageSpecimen holder: Cryo Specimen Holder / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spire / Number images used: 48000
Final two d classificationNumber classes: 2

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