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- EMDB-1072: Three-dimensional structures of translating ribosomes by Cryo-EM. -

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Basic information

Entry
Database: EMDB / ID: EMD-1072
TitleThree-dimensional structures of translating ribosomes by Cryo-EM.
Map data3D reconstruction of E. coli ribosome stalled in translation of Green Fluorescent Protein
Sample
  • Sample: E. coli ribosome translating GFP
  • Complex: E. coli 30S subunit
  • Complex: E. coli 50S subunit
  • RNA: P site tRNA
  • RNA: E site tRNA
  • Protein or peptide: Green fluorescent protein
Biological speciesEscherichia coli (E. coli) / Aequorea victoria (jellyfish)
Methodsingle particle reconstruction / cryo EM / Resolution: 16.0 Å
AuthorsGilbert RJC / Fucini P / Connell S / Fuller SD / Nierhaus KH / Robinson CV / Dobson CM / Stuart DI
CitationJournal: Mol Cell / Year: 2004
Title: Three-dimensional structures of translating ribosomes by Cryo-EM.
Authors: Robert J C Gilbert / Paola Fucini / Sean Connell / Stephen D Fuller / Knud H Nierhaus / Carol V Robinson / Christopher M Dobson / David I Stuart /
Abstract: Cryo-electron microscopy and image reconstruction techniques have been used to obtain three-dimensional maps for E. coli ribosomes stalled following translation of three representative proteins. ...Cryo-electron microscopy and image reconstruction techniques have been used to obtain three-dimensional maps for E. coli ribosomes stalled following translation of three representative proteins. Comparisons of these electron density maps, at resolutions of between 13 and 16 A, with that of a nontranslating ribosome pinpoint specific structural differences in stalled ribosomes and identify additional material, including tRNAs and mRNA. In addition, the tunnel through the large subunit, the anticipated exit route of newly synthesized proteins, is partially occluded in all the stalled ribosome structures. This observation suggests that significant segments of the nascent polypeptide chains examined here could be located within an expanded tunnel, perhaps in a rudimentary globular conformation. Such behavior could be an important aspect of the folding of at least some proteins in the cellular environment.
History
DepositionMar 14, 2004-
Header (metadata) releaseMar 14, 2004-
Map releaseApr 28, 2004-
UpdateOct 31, 2012-
Current statusOct 31, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.154807002
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 1.154807002
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1072.gif

Downloads & links

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Map

FileDownload / File: emd_1072.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of E. coli ribosome stalled in translation of Green Fluorescent Protein
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesY (Sec.)X (Row.)Z (Col.)
3.33 Å/pix.
x 128 pix.
= 426.24 Å		3.33 Å/pix.
x 128 pix.
= 426.24 Å		3.33 Å/pix.
x 128 pix.
= 426.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.33 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 1.06 / Movie #1: 1.154807
Minimum - Maximum-1.96596 - 3.05542
Average (Standard dev.)0.00000000025867 (±0.422637)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZXY
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 426.24 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.333.333.33
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z426.240426.240426.240
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ128128128
MAP C/R/S312
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-1.9663.0550.000

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Supplemental data

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Sample components

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Entire : E. coli ribosome translating GFP

EntireName: E. coli ribosome translating GFP
Components
  • Sample: E. coli ribosome translating GFP
  • Complex: E. coli 30S subunit
  • Complex: E. coli 50S subunit
  • RNA: P site tRNA
  • RNA: E site tRNA
  • Protein or peptide: Green fluorescent protein

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Supramolecule #1000: E. coli ribosome translating GFP

SupramoleculeName: E. coli ribosome translating GFP / type: sample / ID: 1000 / Details: The sample was monodisperse.
Oligomeric state: 30S subunit and 50S subunit, 2 tRNA molecules and 1 nascent protein
Number unique components: 5

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Supramolecule #1: E. coli 30S subunit

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

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Supramolecule #2: E. coli 50S subunit

SupramoleculeName: E. coli 50S subunit / type: complex / ID: 2 / Name.synonym: 50S / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: LSU 50S
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: P site tRNA

MacromoleculeName: P site tRNA / type: rna / ID: 1 / Details: partial occupancy / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #2: E site tRNA

MacromoleculeName: E site tRNA / type: rna / ID: 2 / Details: partial occupancy lower than P site / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #3: Green fluorescent protein

MacromoleculeName: Green fluorescent protein / type: protein_or_peptide / ID: 3 / Name.synonym: GFP / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Aequorea victoria (jellyfish)
Recombinant expressionOrganism: In vitro transcription.translation system / Recombinant plasmid: pIVEX2.3

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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

Concentration5.0 mg/mL
BufferDetails: 20 mM HEPES, 150 mM ammonium acetate, 6 mM magnesium acetate, 2 mM spermidine, 0.05 mM spermine and 4 mM 2-mercaptoethanol. Concentration of ribosomes expressed to A260 units.
GridDetails: 300 mesh copper grid with holey carbon film
VitrificationCryogen name: ETHANE / Chamber temperature: 100 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: Standard unmodified guillotine plunger

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
TemperatureAverage: 100 K
DateMay 2, 2000
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 8.322 µm / Number real images: 10 / Details: Scanner model:UMAX PowerLook 3000 / Od range: 5 / Bits/pixel: 8
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Cs: 2 mm / Nominal defocus max: 5.96 µm / Nominal defocus min: 1.805 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: Each negative dataset
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER, IMAGIC, GAP, CNS, XPLOR
Details: Final maps were calculated from 2276 images from a total of 4500 from 10 individual datasets, with scaling in reciprocal space to crystallographic ribosome structures and correction for map ...Details: Final maps were calculated from 2276 images from a total of 4500 from 10 individual datasets, with scaling in reciprocal space to crystallographic ribosome structures and correction for map anisotropy by B-factor weighting of amplitudes in XPLOR with respect to a similarly-treated control inactive ribosome. Selection of particles for inclusion in the final maps was by correlation coefficient with respect to the alignment model, designed to maximise nascent chain occupancy in the selected images.
Number images used: 2276
Final angle assignmentDetails: SPIDER euler

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Atomic model buiding 1

SoftwareName: GAP
DetailsProtocol: Rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: R-factor and correlation coefficient

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