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- EMDB-3938: In situ subtomogram average of the Chlamydomonas free 26S proteasome -

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Basic information

Entry
Database: EMDB / ID: EMD-3938
TitleIn situ subtomogram average of the Chlamydomonas free 26S proteasome
Map data
Sample
  • Complex: In situ free 26S proteasome
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 23.81 Å
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
DepositionOct 17, 2017-
Header (metadata) releaseOct 25, 2017-
Map releaseOct 25, 2017-
UpdateJan 3, 2018-
Current statusJan 3, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.255
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.255
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3938.png

Downloads & links

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Map

FileDownload / File: emd_3938.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.255 / Movie #1: 0.255
Minimum - Maximum-0.17855318 - 0.76447254
Average (Standard dev.)-0.0013542046 (±0.065478876)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 547.2 Å
α=β=γ: 90.0 °

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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.1790.764-0.001

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Supplemental data

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Sample components

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Entire : In situ free 26S proteasome

EntireName: In situ free 26S proteasome
Components
  • Complex: In situ free 26S proteasome

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Supramolecule #1: In situ free 26S proteasome

SupramoleculeName: In situ free 26S proteasome / type: complex / ID: 1 / Parent: 0
Details: In situ subtomogram average generated from free 26S proteasomes imaged within the native Chlamydomonas cell. Cells were thinned by focused ion beam milling.
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Strain: mat3-4
Molecular weightTheoretical: 2 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7
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..
DetailsFree 26S proteasome within the nucleus and cytoplasm of the native Chlamydomonas cell. Whole cells were plunge-frozen onto EM grids and then thinned with a cryo-focused ion beam instrument.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 4.0 µm / Nominal magnification: 42000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 23.81 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION
Details: For the final reconstruction, the tilt series was restricted between -30 and +30 degrees.
Number subtomograms used: 2116
ExtractionNumber tomograms: 76 / Number images used: 3385
Reference model: EMDB: 2165, lowpass filtered, one masked cap
Method: automated template matching / Software - Name: PyTom / Software - details: template matching
Details: Extracted proteasomes were cut in half along the medial axis of the 20S core particle, treating each half as an individual particle.
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Software - details: CTFFIND was used with the Relion package
Final 3D classificationSoftware - Name: PyTom
Software - details: We used several rounds of autofocused 3D classification (AC3D)
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION

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