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- EMDB-5225: Human 80S ribosome in situ, puromycin-treated -

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Basic information

Entry
Database: EMDB / ID: EMD-5225
TitleHuman 80S ribosome in situ, puromycin-treated
Map dataThis is a map of a tomographic average of a human 80S ribosome in situ treated with puromycin
Sample
  • Sample: Human 80S ribosome in situ, puromycin-treated
  • Complex: cytosolic 80S ribosome
Keywordshuman 80S ribosome / puromycin / in situ / cytosol / polysome / polyribosome / protein synthesis / translation / 3D cryoEM / tomography / cellular tomography
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / negative staining / Resolution: 39.0 Å
AuthorsBrandt F / Carlson L-A / Hartl FU / Baumeister W / Grunewald K
CitationJournal: Mol Cell / Year: 2010
Title: The three-dimensional organization of polyribosomes in intact human cells.
Authors: Florian Brandt / Lars-Anders Carlson / F Ulrich Hartl / Wolfgang Baumeister / Kay Grünewald /
Abstract: Structural studies have provided detailed insights into different functional states of the ribosome and its interaction with factors involved in nascent peptide folding, processing, and targeting. ...Structural studies have provided detailed insights into different functional states of the ribosome and its interaction with factors involved in nascent peptide folding, processing, and targeting. However, how the translational machinery is organized spatially in native cellular environments is not yet well understood. Here we have mapped individual ribosomes in electron tomograms of intact human cells by template matching and determined the average structure of the ribosome in situ. Characteristic features of active ribosomes in the cellular environment were assigned to the tRNA channel, elongation factors, and additional densities near the peptide tunnel. Importantly, the relative spatial configuration of neighboring ribosomes in the cell is clearly nonrandom. The preferred configurations are specific for active polysomes and were largely abrogated in puromycin-treated control cells. The distinct neighbor orientations found in situ resemble configurations of bacterial polysomes in vitro, indicating a conserved supramolecular organization with implications for nascent polypeptide folding.
History
DepositionAug 10, 2010-
Header (metadata) releaseDec 9, 2010-
Map releaseDec 9, 2010-
UpdateMar 6, 2013-
Current statusMar 6, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.9
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.9
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5225_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5225.map.gz / Format: CCP4 / Size: 422.9 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a map of a tomographic average of a human 80S ribosome in situ treated with puromycin
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
8.21 Å/pix.
x 48 pix.
= 394.08 Å		8.21 Å/pix.
x 48 pix.
= 394.08 Å		8.21 Å/pix.
x 48 pix.
= 394.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 8.21 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.9 / Movie #1: 0.9
Minimum - Maximum-1.913486 - 4.19849586
Average (Standard dev.)0.0 (±0.99999547)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions484848
Spacing484848
CellA=B=C: 394.08002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z8.218.218.21
M x/y/z484848
origin x/y/z0.0000.0000.000
length x/y/z394.080394.080394.080
α/β/γ90.00090.00090.000
start NX/NY/NZ-99-99-99
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS484848
D min/max/mean-1.9134.198-0.000

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Supplemental data

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Sample components

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Entire : Human 80S ribosome in situ, puromycin-treated

EntireName: Human 80S ribosome in situ, puromycin-treated
Components
  • Sample: Human 80S ribosome in situ, puromycin-treated
  • Complex: cytosolic 80S ribosome

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Supramolecule #1000: Human 80S ribosome in situ, puromycin-treated

SupramoleculeName: Human 80S ribosome in situ, puromycin-treated / type: sample / ID: 1000 / Details: Cytosolic ribosomes in situ / Oligomeric state: One 80S ribosome within cytosol / Number unique components: 1
Molecular weightMethod: Sedimentation, 80S

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Supramolecule #1: cytosolic 80S ribosome

SupramoleculeName: cytosolic 80S ribosome / type: complex / ID: 1 / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Homo sapiens (human) / synonym: human

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Details: Cellular medium, DMEM (Gibco) supplemented with 10% foetal calf serum, 37C and 5% CO2
StainingType: NEGATIVE / Details: Cells grown on grids, vitrification
GridDetails: C-flat 2/1, holey carbon gold grid
VitrificationCryogen name: ETHANE / Chamber temperature: 77 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: plunger. Vitrification carried out in air
Method: Blot for 2 s before plunging

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Electron microscopy

MicroscopeFEI/PHILIPS CM300FEG/T
TemperatureMin: 77 K / Max: 77 K / Average: 77 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 50,000 times magnification
Specialist opticsEnergy filter - Name: GIF2002 / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
DateDec 3, 2008
Image recordingCategory: CCD / Film or detector model: GATAN MULTISCAN / Average electron dose: 80 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 17500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 6.0 µm / Nominal magnification: 17500
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder.
Specimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -65 ° / Tilt series - Axis1 - Max angle: 65 °

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Image processing

DetailsIndividual particle subvolumes were automatically selected by CCC threshold.
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 39.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EM / Details: exact weighting / Number subtomograms used: 1568
Final 3D classificationNumber classes: 1

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