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- EMDB-1582: Three-dimensional structure of the 70S E. coli ribosome in its na... -

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Basic information

Entry
Database: EMDB / ID: EMD-1582
TitleThree-dimensional structure of the 70S E. coli ribosome in its native 3D organization in polysomes.
Map dataThis is a 3D density of class t-t ribosomes obtained by averaging of polysomal particles.
Sample
  • Sample: 70S ribosome in a native polysome with a neighbor ribosome at the 5' end oriented in a tt-classRibosome
  • Complex: 70S ribosomeRibosome
Keywords70S ribosome / polysome / translation / protein folding / cryoelectron tomography
Biological speciesEscherichia coli (E. coli)
Methodsubtomogram averaging / cryo EM / Resolution: 30.0 Å
AuthorsBrandt F / Elcock AH / Etchells SA / Ortiz JO / Hartl FU / Baumeister W
CitationJournal: Cell / Year: 2009
Title: The native 3D organization of bacterial polysomes.
Authors: Florian Brandt / Stephanie A Etchells / Julio O Ortiz / Adrian H Elcock / F Ulrich Hartl / Wolfgang Baumeister /
Abstract: Recent advances have led to insights into the structure of the bacterial ribosome, but little is known about the 3D organization of ribosomes in the context of translating polysomes. We employed ...Recent advances have led to insights into the structure of the bacterial ribosome, but little is known about the 3D organization of ribosomes in the context of translating polysomes. We employed cryoelectron tomography and a template-matching approach to map 70S ribosomes in vitrified bacterial translation extracts and in lysates of active E. coli spheroplasts. In these preparations, polysomal arrangements were observed in which neighboring ribosomes are densely packed and exhibit preferred orientations. Analysis of characteristic examples of polysomes reveals a staggered or pseudohelical organization of ribosomes along the mRNA trace, with the transcript being sequestered on the inside, the tRNA entrance sites being accessible, and the polypeptide exit sites facing the cytosol. Modeling of elongating nascent polypeptide chains suggests that this arrangement maximizes the distance between nascent chains on adjacent ribosomes, thereby reducing the probability of intermolecular interactions that would give rise to aggregation and limit productive folding.
History
DepositionNov 7, 2008-
Header (metadata) releaseNov 10, 2008-
Map releaseJun 11, 2009-
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: 9.0E-9
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 9.0E-9
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1582.gif

Downloads & links

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Map

FileDownload / File: emd_1582.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a 3D density of class t-t ribosomes obtained by averaging of polysomal particles.
Voxel sizeX=Y=Z: 5.6 Å
Density
Contour LevelBy EMDB: 0.0000000092
Minimum - Maximum-0.0000000952641 - 0.000000164415
Average (Standard dev.)-0.00000000720858 (±0.0000000130023)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 716.8 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.65.65.6
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z716.800716.800716.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-100-100-99
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0000.000-0.000

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Supplemental data

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Sample components

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Entire : 70S ribosome in a native polysome with a neighbor ribosome at the...

EntireName: 70S ribosome in a native polysome with a neighbor ribosome at the 5' end oriented in a tt-classRibosome
Components
  • Sample: 70S ribosome in a native polysome with a neighbor ribosome at the 5' end oriented in a tt-classRibosome
  • Complex: 70S ribosomeRibosome

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Supramolecule #1000: 70S ribosome in a native polysome with a neighbor ribosome at the...

SupramoleculeName: 70S ribosome in a native polysome with a neighbor ribosome at the 5' end oriented in a tt-class
type: sample / ID: 1000
Details: The sample is indeed heterogeneous, a prokaryotic lysate containing many cellular components
Oligomeric state: ribosomes forming polysomes / Number unique components: 1
Molecular weightExperimental: 2.7 MDa / Theoretical: 2.7 MDa / Method: Sedimentation

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Supramolecule #1: 70S ribosome

SupramoleculeName: 70S ribosome / type: complex / ID: 1 / Name.synonym: 70S ribosome
Details: The ribosomes under scrutiny are part of large polysomal arrangements
Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightExperimental: 2.7 MDa / Theoretical: 2.7 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration20 mg/mL
BufferpH: 7.5
Details: Roche reconstitution buffer from the RTS kit, ATP,GTP, 8-15 mM Mg2, 100-250 nm K,NH4,DTT, protease inhibitors,NaN3
GridDetails: 400 mesh gold grid
VitrificationCryogen name: ETHANE / Chamber temperature: 77 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: plunger. Vitrification carried out in air
Method: Blot for 1 s before plunging

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 160 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 53960 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 27500
Sample stageSpecimen holder: Side entry liquid nitrogen- / Specimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
TemperatureMin: 70 K / Max: 90 K / Average: 77 K
Alignment procedureLegacy - Astigmatism: Objective astigmatism was corrected using a quadrupole stigmator at 50,000 times magnification
Legacy - Electron beam tilt params: -4
DateDec 1, 2006
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS (4k x 4k) / Average electron dose: 50 e/Å2

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Image processing

Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 30.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: TOM ToolBox / Details: CET
DetailsAverage number of projections used in the 3D reconstructions: 391.

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Atomic model buiding 1

Initial modelPDB ID:

2aw7
PDB Unreleased entry

SoftwareName: Chimera
DetailsProtocol: Rigid Body
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

2awb
PDB Unreleased entry

SoftwareName: Chimera
DetailsProtocol: Rigid Body
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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