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- EMDB-1394: Snapshots of nuclear pore complexes in action captured by cryo-el... -

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Basic information

Entry
Database: EMDB / ID: EMD-1394
TitleSnapshots of nuclear pore complexes in action captured by cryo-electron tomography.
Map dataasymmetric unit of Dictyostelium discoideum nuclear pore complex
Sample
  • Sample: nuclear pore complex
  • Organelle or cellular component: nuclear pore
Biological speciesDictyostelium discoideum (eukaryote)
Methodsubtomogram averaging / cryo EM / Resolution: 58.0 Å
AuthorsBeck M / Lucic V / Forster F / Baumeister W / Medalia O
CitationJournal: Nature / Year: 2007
Title: Snapshots of nuclear pore complexes in action captured by cryo-electron tomography.
Authors: Martin Beck / Vladan Lucić / Friedrich Förster / Wolfgang Baumeister / Ohad Medalia /
Abstract: Nuclear pore complexes reside in the nuclear envelope of eukaryotic cells and mediate the nucleocytoplasmic exchange of macromolecules. Traffic is regulated by mobile transport receptors that target ...Nuclear pore complexes reside in the nuclear envelope of eukaryotic cells and mediate the nucleocytoplasmic exchange of macromolecules. Traffic is regulated by mobile transport receptors that target their cargo to the central translocation channel, where phenylalanine-glycine-rich repeats serve as binding sites. The structural analysis of the nuclear pore is a formidable challenge given its size, its location in a membranous environment and its dynamic nature. Here we have used cryo-electron tomography to study the structure of nuclear pore complexes in their functional environment, that is, in intact nuclei of Dictyostelium discoideum. A new image-processing strategy compensating for deviations of the asymmetric units (protomers) from a perfect eight-fold symmetry enabled us to refine the structure and to identify new features. Furthermore, the superposition of a large number of tomograms taken in the presence of cargo, which was rendered visible by gold nanoparticles, has yielded a map outlining the trajectories of import cargo. Finally, we have performed single-molecule Monte Carlo simulations of nuclear import to interpret the experimentally observed cargo distribution in the light of existing models for nuclear import.
History
DepositionJul 25, 2007-
Header (metadata) releaseJul 25, 2007-
Map releaseAug 5, 2011-
UpdateAug 5, 2011-
Current statusAug 5, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 165.731958763
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 165.731958763
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-1394.png

Downloads & links

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Map

FileDownload / File: emd_1394.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationasymmetric unit of Dictyostelium discoideum nuclear pore complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
16.4 Å/pix.
x 128 pix.
= 2099.2 Å		16.4 Å/pix.
x 128 pix.
= 2099.2 Å		16.4 Å/pix.
x 128 pix.
= 2099.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 16.4 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 200.0 / Movie #1: 165.7319588
Minimum - Maximum0.0 - 255.0
Average (Standard dev.)127.192999999999998 (±35.792499999999997)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 2099.2 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z16.416.416.4
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z2099.2002099.2002099.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean0.000255.000127.193

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Supplemental data

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Sample components

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Entire : nuclear pore complex

EntireName: nuclear pore complex
Components
  • Sample: nuclear pore complex
  • Organelle or cellular component: nuclear pore

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Supramolecule #1000: nuclear pore complex

SupramoleculeName: nuclear pore complex / type: sample / ID: 1000
Oligomeric state: octamer (actual refined structure represents an asymmetric unit of the NPC)
Number unique components: 1

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Supramolecule #1: nuclear pore

SupramoleculeName: nuclear pore / type: organelle_or_cellular_component / ID: 1 / Name.synonym: nuclear pore / Oligomeric state: octamer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Dictyostelium discoideum (eukaryote) / Strain: AX2 / Organelle: Nucleus / Location in cell: nuclear enveope

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6
Details: 50 mM Tris pH 7.6, 25 mM KCl, 5mM MgCl2, 50 mM sucrose
GridDetails: 200 mesh copper grid
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: Rudolf Gatz plunger

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Electron microscopy

MicroscopeFEI TECNAI F30
TemperatureAverage: 90 K
Specialist opticsEnergy filter - Name: GIF2002 / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Image recordingCategory: CCD / Film or detector model: GENERIC CCD
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 27500
Sample stageSpecimen holder: Eucentric / Specimen holder model: OTHER / Tilt series - Axis1 - Min angle: -63 ° / Tilt series - Axis1 - Max angle: 63 °
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

DetailsAverage number of projections used in the 3D reconstructions: 4184.
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 58.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: TOM

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