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- EMDB-1073: Three-dimensional structures of translating ribosomes by Cryo-EM. -

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Basic information

Entry
Database: EMDB / ID: EMD-1073
TitleThree-dimensional structures of translating ribosomes by Cryo-EM.
Map dataAverage difference map calculated between individual difference maps for Ig2 and GFP ribosome-nascent chain complexes (see entries 1068, 1070, 1071, 1072).
Sample
  • Sample: E. coli ribosome translating GFP and tandem Ig domains - difference map
  • RNA: P site tRNA
  • RNA: E site tRNA
  • Protein or peptide: nascent protein
Biological speciesEscherichia coli (E. coli) / Dictyostelium discoideum (eukaryote)
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 15, 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: 162.883841256
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 162.883841256
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1073.gif

Downloads & links

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Map

FileDownload / File: emd_1073.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAverage difference map calculated between individual difference maps for Ig2 and GFP ribosome-nascent chain complexes (see entries 1068, 1070, 1071, 1072).
Voxel sizeX=Y=Z: 3.33 Å
Density
Contour Level1: 65.200000000000003 / Movie #1: 162.8838413
Minimum - Maximum-319.889999999999986 - 325.819999999999993
Average (Standard dev.)-0.000938282 (±26.123799999999999)
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-319.890325.820-0.001

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Supplemental data

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Sample components

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Entire : E. coli ribosome translating GFP and tandem Ig domains - differen...

EntireName: E. coli ribosome translating GFP and tandem Ig domains - difference map
Components
  • Sample: E. coli ribosome translating GFP and tandem Ig domains - difference map
  • RNA: P site tRNA
  • RNA: E site tRNA
  • Protein or peptide: nascent protein

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Supramolecule #1000: E. coli ribosome translating GFP and tandem Ig domains - differen...

SupramoleculeName: E. coli ribosome translating GFP and tandem Ig domains - difference map
type: sample / ID: 1000 / Details: The sample was monodisperse.
Oligomeric state: 2 tRNA molecules and the average of two nascent proteins
Number unique components: 3

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Macromolecule #1: P site tRNA

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

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Macromolecule #3: nascent protein

MacromoleculeName: nascent protein / type: protein_or_peptide / ID: 3 / Name.synonym: GFP-Ig2
Details: Density for nascent protein is average of that for tandem Ig domains and GFP
Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Dictyostelium discoideum (eukaryote)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pT7-7

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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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Cs: 2 mm / Nominal magnification: 50000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN
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 / Details: Scanner model: UMAX PowerLook 3000 / Od range: 5 / Bits/pixel: 8

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Image processing

CTF correctionDetails: Each negative dataset
Final angle assignmentDetails: SPIDER euler
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: See entries 1068, 1070, 1071 and 1072. Vector difference maps were calculated between reconstructions of each nascent protein-ribosome complex and the control inactive map. The difference ...Details: See entries 1068, 1070, 1071 and 1072. Vector difference maps were calculated between reconstructions of each nascent protein-ribosome complex and the control inactive map. The difference densities for the tandem Ig domains and GFP nascent proteins were more similar to each other than either was to the single Ig domain nascent protein. Therefore these two difference maps were averaged together to produce a set of consensus difference densities representing the main differences between inactive ribosomes and ribosomes stalled during translation that contain a nascent polypeptide chain.

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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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