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- EMDB-3967: In situ cryo-electron tomogram from Chlamydomonas reinhardtii of ... -

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Entry
Database: EMDB / ID: 3967
TitleIn situ cryo-electron tomogram from Chlamydomonas reinhardtii of the cellular environment around the nuclear envelope
Map dataCryo-electron tomogram of Chlamydomonas reinhardtii nuclear membrane
SampleWhole Chlamydomonas cells:
SourceChlamydomonas reinhardtii (plant)
Methodelectron tomography / cryo EM
AuthorsAlbert S / Schaffer M / Beck F / Mosalaganti S / Asano S / Thomas HF / Plitzko J / Beck M / Baumeister W / Engel BD
CitationJournal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2017
Title: Proteasomes tether to two distinct sites at the nuclear pore complex.
Authors: Sahradha Albert / Miroslava Schaffer / Florian Beck / Shyamal Mosalaganti / Shoh Asano / Henry F Thomas / Jürgen M Plitzko / Martin Beck / Wolfgang Baumeister / Benjamin D Engel
Abstract: The partitioning of cellular components between the nucleus and cytoplasm is the defining feature of eukaryotic life. The nuclear pore complex (NPC) selectively gates the transport of macromolecules ...The partitioning of cellular components between the nucleus and cytoplasm is the defining feature of eukaryotic life. The nuclear pore complex (NPC) selectively gates the transport of macromolecules between these compartments, but it is unknown whether surveillance mechanisms exist to reinforce this function. By leveraging in situ cryo-electron tomography to image the native cellular environment of , we observed that nuclear 26S proteasomes crowd around NPCs. Through a combination of subtomogram averaging and nanometer-precision localization, we identified two classes of proteasomes tethered via their Rpn9 subunits to two specific NPC locations: binding sites on the NPC basket that reflect its eightfold symmetry and more abundant binding sites at the inner nuclear membrane that encircle the NPC. These basket-tethered and membrane-tethered proteasomes, which have similar substrate-processing state frequencies as proteasomes elsewhere in the cell, are ideally positioned to regulate transcription and perform quality control of both soluble and membrane proteins transiting the NPC.
DateDeposition: Nov 7, 2017 / Header (metadata) release: Nov 15, 2017 / Map release: Nov 22, 2017 / Last update: Jan 3, 2018

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Structure visualization

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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Simplified surface model
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Structure viewerEM map:
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Supplemental images

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Map

Fileemd_3967.map.gz (map file in CCP4 format, 799179 KB)
Projections & slices

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Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
464 pix
13.68 Å/pix.
= 6347.52 Å
928 pix
13.68 Å/pix.
= 12695.04 Å
928 pix
13.68 Å/pix.
= 12695.04 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 13.68 Å
Density
Contour Level:15 (by author)
Minimum - Maximum-112 - 109
Average (Standard dev.)2.7339582 (10.788662)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions928928464
Origin00232
Limit927927695
Spacing928928464
CellA: 12695.04 Å / B: 12695.04 Å / C: 6347.52 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z13.6813.6813.68
M x/y/z928928464
origin x/y/z0.0000.0000.000
length x/y/z12695.04012695.0406347.520
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS00232
NC/NR/NS928928464
D min/max/mean-112.000109.0002.734

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Supplemental data

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Sample components

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Entire Whole Chlamydomonas cells

EntireName: Whole Chlamydomonas cells
Details: Grown suspended in TAP media, with normal atmosphere aeration and constant light
Number of components: 1

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Component #1: cellular-component, Whole Chlamydomonas cells

Cellular-componentName: Whole Chlamydomonas cells
Details: Grown suspended in TAP media, with normal atmosphere aeration and constant light
Recombinant expression: No
SourceSpecies: Chlamydomonas reinhardtii (plant) / Strain: mat3-4

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Experimental details

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Sample preparation

SpecimenSpecimen state: cell / Method: cryo EM
Sample solutionBuffer solution: TAP media / pH: 7
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE MIXTURE / Temperature: 293 K / Humidity: 90 %
Details: Blotted for 10 seconds with 10 blot force before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.5 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 42000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 4000 - 6000 nm / Energy filter: GIF Quantum LS / Energy window: 0-20 eV
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt Angle: -60 - 60 deg.
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionDetails: Images were collected in movie mode at 12 frames per second

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Image processing

ProcessingMethod: electron tomography / Tilt angle increment: 2 deg. / Number of sections: 61
3D reconstructionAlgorithm: BACK PROJECTION / Software: IMOD / Details: This is a bin4 (twice binned) tomogram.

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