Journal: 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
Deposition
Oct 17, 2017
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Header (metadata) release
Oct 25, 2017
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Map release
Oct 25, 2017
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Update
Dec 25, 2019
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Current status
Dec 25, 2019
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_3935.map.gz / Format: CCP4 / Size: 3.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel size
X=Y=Z: 3.42 Å
Density
Contour Level
By AUTHOR: 0.255 / Movie #1: 0.255
Minimum - Maximum
-0.24600284 - 0.85752934
Average (Standard dev.)
0.025231246 (±0.14279063)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
96
96
96
Spacing
96
96
96
Cell
A=B=C: 328.32 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
3.42
3.42
3.42
M x/y/z
96
96
96
origin x/y/z
0.000
0.000
0.000
length x/y/z
328.320
328.320
328.320
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
-200
-200
-200
NX/NY/NZ
401
401
401
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
96
96
96
D min/max/mean
-0.246
0.858
0.025
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Supplemental data
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Sample components
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Entire : In situ processing state 26S proteasome
Entire
Name: In situ processing state 26S proteasome
Components
Complex: In situ processing state 26S proteasome
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Supramolecule #1: In situ processing state 26S proteasome
Supramolecule
Name: In situ processing state 26S proteasome / type: complex / ID: 1 / Parent: 0 Details: In situ subtomogram average generated from processing state 26S proteasomes imaged within the native Chlamydomonas cell. Cells were thinned by focused ion beam milling.
Cryogen 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..
Details
Processing state 26S proteasome within the cytoplasm and nucleus 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
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Image recording
Film 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 beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 19.61 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: 1156
Extraction
Number tomograms: 76 / Number images used: 5498 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 correction
Software - Name: CTFFIND (ver. 4) Software - details: CTFFIND was used with the Relion package
Final 3D classification
Software - Name: PyTom Software - details: We used several rounds of autofocused 3D classification (AC3D)
Final angle assignment
Type: NOT APPLICABLE / Software - Name: RELION
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