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- EMDB-5226: Human 80S ribosomes within a helical polysome in a cellular subto... -

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Basic information

Entry
Database: EMDB / ID: EMD-5226
TitleHuman 80S ribosomes within a helical polysome in a cellular subtomogram
Map dataThis is a tomographic subvolume containing a helical polysome in the cytosol of a human cell
Sample
  • Sample: Human helical polysome, subtomogram of cytosol
  • Organelle or cellular component: Cytosol
Keywordshuman 80S ribosome / puromycin / in situ / cytosol / polysome / polyribosome / protein synthesis / translation / 3D cryoEM / tomography / cellular tomography
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM / negative staining
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-
UpdateNov 16, 2011-
Current statusNov 16, 2011Processing site: RCSB / Status: Released

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Structure visualization

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Supplemental images

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Map

FileDownload / File: emd_5226.map.gz / Format: CCP4 / Size: 5.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a tomographic subvolume containing a helical polysome in the cytosol of a human cell
Voxel sizeX=Y=Z: 16.4 Å
Density
Contour LevelBy AUTHOR: 0.0
Minimum - Maximum-2.36651 - 2.36651
Average (Standard dev.)-0.000190069 (±0.787292)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-72-72-36
Dimensions14414472
Spacing14414472
CellA: 2361.6 Å / B: 2361.6 Å / C: 1180.8 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z16.416.416.4
M x/y/z14414472
origin x/y/z0.0000.0000.000
length x/y/z2361.6002361.6001180.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-99-99-99
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-72-72-36
NC/NR/NS14414472
D min/max/mean-2.3672.367-0.000

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Supplemental data

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Sample components

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Entire : Human helical polysome, subtomogram of cytosol

EntireName: Human helical polysome, subtomogram of cytosol
Components
  • Sample: Human helical polysome, subtomogram of cytosol
  • Organelle or cellular component: Cytosol

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Supramolecule #1000: Human helical polysome, subtomogram of cytosol

SupramoleculeName: Human helical polysome, subtomogram of cytosol / type: sample / ID: 1000 / Details: Cellular ribosomes in situ / Oligomeric state: ca. 15-20 ribosomes within a polysome / Number unique components: 1

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Supramolecule #1: Cytosol

SupramoleculeName: Cytosol / type: organelle_or_cellular_component / ID: 1 / Name.synonym: Polysomal ribosomes / Details: Ca. 15-20 ribosomes within a cytosolic polysome / Number of copies: 15 / Oligomeric state: polysome / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: U87MG / Organelle: Cytosol / Location in cell: cytosolic

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingelectron tomography

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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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 17500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 6.0 µm / Nominal magnification: 17500
Specialist opticsEnergy filter - Name: GIF2002 / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
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 ° / Tilt series - Axis1 - Angle increment: 2 °
TemperatureMin: 77 K / Max: 77 K / Average: 77 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 50,000 times magnification
DateJul 3, 2008
Image recordingCategory: CCD / Film or detector model: GATAN MULTISCAN / Average electron dose: 80 e/Å2

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Image processing

Final reconstructionAlgorithm: OTHER / Software - Name: EM / Details: exact weighting / Number images used: 72
DetailsIndividual particle subvolumes were automatically selected by CCC threshold

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