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- EMDB-3936: In situ subtomogram average of the Chlamydomonas membrane-tethere... -

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Basic information

Entry
Database: EMDB / ID: 3936
TitleIn situ subtomogram average of the Chlamydomonas membrane-tethered 26S proteasome
Map data
SampleIn situ membrane-tethered 26S proteasome:
SourceChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / 24.87 Å resolution
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: Oct 17, 2017 / Header (metadata) release: Oct 25, 2017 / Map release: Oct 25, 2017 / Last update: Jan 3, 2018

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Structure visualization

Movie
  • Surface view colored by radius
  • Surface level: 0.175
  • Imaged by UCSF Chimera
  • Download
  • Surface view with section colored by density value
  • Surface level: 0.175
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_3936.map.gz (map file in CCP4 format, 16385 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
160 pix
3.42 Å/pix.
= 547.2 Å
160 pix
3.42 Å/pix.
= 547.2 Å
160 pix
3.42 Å/pix.
= 547.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.42 Å
Density
Contour Level:0.175 (by author), 0.175 (movie #1):
Minimum - Maximum-0.24740675 - 0.7397931
Average (Standard dev.)0.005593523 (0.07845841)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions160160160
Origin000
Limit159159159
Spacing160160160
CellA=B=C: 547.2 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.423.423.42
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z547.200547.200547.200
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.2470.7400.006

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Supplemental data

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Sample components

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Entire In situ membrane-tethered 26S proteasome

EntireName: In situ membrane-tethered 26S proteasome
Details: In situ subtomogram average generated from membrane-tethered 26S proteasomes imaged within the native Chlamydomonas cell. Cells were thinned by focused ion beam milling.
Number of components: 1
MassTheoretical: 2 MDa

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Component #1: protein, In situ membrane-tethered 26S proteasome

ProteinName: In situ membrane-tethered 26S proteasome
Details: In situ subtomogram average generated from membrane-tethered 26S proteasomes imaged within the native Chlamydomonas cell. Cells were thinned by focused ion beam milling.
Recombinant expression: No
MassTheoretical: 2 MDa
SourceSpecies: Chlamydomonas reinhardtii (plant) / Strain: mat3-4
Source (natural)Organelle: nucleus / Location in cell: inner nuclear membrane

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Experimental details

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Sample preparation

SpecimenSpecimen state: cell / Method: cryo EM
Sample solutionpH: 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: subtomogram averaging / Applied symmetry: C1 (asymmetric)
3D reconstructionAlgorithm: BACK PROJECTION / Software: RELION / Resolution: 24.87 Å / Resolution method: FSC 0.143 CUT-OFF
Details: For the final reconstruction, the tilt series was restricted between -30 and +30 degrees.

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