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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 data
SampleE. coli ribosome translating GFP:
(ribosome-prokaryote) x 2 / (nucleic-acidNucleic acid) x 2 / Green fluorescent protein
Biological speciesEscherichia coli (E. coli) / Aequorea victoria (jellyfish)
Methodsingle particle reconstruction / cryo EM / Resolution: 16 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
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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)
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
Contour LevelPrimary: 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 / Details: The sample was monodisperse.
Oligomeric State: 30S subunit and 50S subunit, 2 tRNA molecules and 1 nascent protein
Number of components: 5

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Component #1: ribosome-prokaryote, E. coli 30S subunit

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

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Component #2: ribosome-prokaryote, E. coli 50S subunit

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

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Component #3: nucleic-acid, P site tRNA

nucleic acidName: P site tRNA / Class: RNA / Details: partial occupancy / Structure: SINGLE STRANDED / Synthetic: No
SourceSpecies: Escherichia coli (E. coli)

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Component #4: nucleic-acid, E site tRNA

nucleic acidName: E site tRNA / Class: RNA / Details: partial occupancy lower than P site / Structure: SINGLE STRANDED / Synthetic: No
SourceSpecies: Escherichia coli (E. coli)

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Component #5: protein, Green fluorescent protein

ProteinName: Green fluorescent protein / a.k.a: GFP / Oligomeric Details: Monomer / Number of Copies: 1 / Recombinant expression: Yes
SourceSpecies: Aequorea victoria (jellyfish)
Source (engineered)Expression System: In vitro transcription.translation system
Vector: pIVEX2.3

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 5 mg/mL
Buffer solution: 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.
Support film300 mesh copper grid with holey carbon film
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 100 K
Details: Vitrification instrument: Standard unmodified guillotine plunger

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Electron microscopy imaging

ImagingMicroscope: FEI/PHILIPS CM200FEG / Date: May 2, 2000
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
LensMagnification: 50000 X (nominal) / Cs: 2 mm / Imaging mode: OTHER / Defocus: 1805 - 5960 nm
Specimen HolderHolder: Eucentric / Model: GATAN LIQUID NITROGEN / Temperature: 100
CameraDetector: KODAK SO-163 FILM

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Image acquisition

Image acquisitionNumber of digital images: 10 / Scanner: OTHER / Sampling size: 8.322 µm / Bit depth: 8 / OD range: 5 / Details: Scanner model:UMAX PowerLook 3000

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 2276 / Applied symmetry: C1 (asymmetric)
3D reconstructionAlgorithm: Projection matching / Software: SPIDER, IMAGIC, GAP, CNS, XPLOR / CTF correction: Each negative dataset / Resolution: 16 Å / Resolution method: FSC 0.5 / Euler angles: SPIDER euler
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.

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Atomic model buiding

Modeling #1Software: GAP / Refinement protocol: rigid body / Target criteria: R-factor and correlation coefficient / Refinement space: REAL / Details: Protocol: Rigid body

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