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- EMDB-3940: Local refinement of the extra density that tethers 26S proteasome... -

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Entry
Database: EMDB / ID: 3940
TitleLocal refinement of the extra density that tethers 26S proteasomes to the inner nuclear membrane
Map data
SampleLocal refinement of the extra density that tethers 26S proteasomes to the inner nuclear membrane:
SourceChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / 27.36 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 with section colored by density value
  • Surface level: 0.175
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.175
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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Map

Fileemd_3940.map.gz (map file in CCP4 format, 33133 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
202 pix
3.42 Å/pix.
= 690.84 Å
202 pix
3.42 Å/pix.
= 690.84 Å
203 pix
3.42 Å/pix.
= 694.26 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-0.20379238 - 1.3181543
Average (Standard dev.)0.0066731554 (0.08231385)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions202203202
Origin-1-1-1
Limit200201200
Spacing203202202
CellA: 694.26 Å / B: 690.84 Å / C: 690.84 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.423.423.42
M x/y/z203202202
origin x/y/z0.0000.0000.000
length x/y/z694.260690.840690.840
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS-1-1-1
NC/NR/NS203202202
D min/max/mean-0.2041.3180.007

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Supplemental data

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Sample components

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Entire Local refinement of the extra density that tethers 26S proteasome...

EntireName: Local refinement of the extra density that tethers 26S proteasomes to the inner nuclear membrane
Details: Fusion structure of two in situ subtomogram averages: the 26S double-capped proteasome (EMDB-3932) and a local refinement of the extra density tethering the proteasome to the inner nuclear membrane (EMDB-3936). Proteasomes were 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, Local refinement of the extra density that tethers 26S p...

ProteinName: Local refinement of the extra density that tethers 26S proteasomes to the inner nuclear membrane
Details: Fusion structure of two in situ subtomogram averages: the 26S double-capped proteasome (EMDB-3932) and a local refinement of the extra density tethering the proteasome to the inner nuclear membrane (EMDB-3936). Proteasomes were 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
3D reconstructionResolution: 27.36 Å / Resolution method: FSC 0.143 CUT-OFF
Details: Fusion of a 26S double-capped proteasome subtomogram average (EMDB-3932) with locally refined extra density from the membrane-tethered 26S proteasome subtomogram average (EMDB-3936)

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