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

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Basic information

Entry
Database: EMDB / ID: EMD-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
Sample
  • Cell: Whole Chlamydomonas cells
Biological speciesChlamydomonas 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.
History
DepositionNov 7, 2017-
Header (metadata) releaseNov 15, 2017-
Map releaseNov 22, 2017-
UpdateNov 28, 2018-
Current statusNov 28, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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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:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3967.png

Downloads & links

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Map

FileDownload / File: emd_3967.map.gz / Format: CCP4 / Size: 762.2 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationCryo-electron tomogram of Chlamydomonas reinhardtii nuclear membrane
Voxel sizeX=Y=Z: 13.68 Å
Density
Minimum - Maximum-112. - 109.
Average (Standard dev.)2.7339582 (±10.788662)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00232
Dimensions928928464
Spacing928928464
CellA: 12695.04 Å / B: 12695.04 Å / C: 6347.52 Å
α=β=γ: 90.0 °

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
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
Components
  • Cell: Whole Chlamydomonas cells

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Supramolecule #1: Whole Chlamydomonas cells

SupramoleculeName: Whole Chlamydomonas cells / type: cell / ID: 1 / Parent: 0
Details: Grown suspended in TAP media, with normal atmosphere aeration and constant light
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Strain: mat3-4

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7 / Details: TAP media
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 90 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV
Details: Blotted for 10 seconds with 10 blot force before plunging..
DetailsThe cells were frozen onto grids, then thinned using cryo-focused ion beam milling.
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.03 nA / Focused ion beam - Dose rate: 2 / Focused ion beam - Duration: 2400 sec. / Focused ion beam - Temperature: 80 K / Focused ion beam - Initial thickness: 5000 / Focused ion beam - Final thickness: 100
Focused ion beam - Details: Starting curent: 0.5 nA, Final current: 0.03 nA. The value given for _emd_sectioning_focused_ion_beam.instrument is FEI Scios DB-FIB. This is not in a list of allowed ...Focused ion beam - Details: Starting curent: 0.5 nA, Final current: 0.03 nA. The value given for _emd_sectioning_focused_ion_beam.instrument is FEI Scios DB-FIB. This is not in a list of allowed values set(['DB235', 'OTHER']) so OTHER is written into the XML file.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 4.0 µm / Nominal magnification: 42000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Average exposure time: 1.5 sec. / Average electron dose: 1.5 e/Å2
Details: Images were collected in movie mode at 12 frames per second
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: IMOD
Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD / Details: This is a bin4 (twice binned) tomogram. / Number images used: 61

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