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- EMDB-12917: Electron cryo-tomogram of T20S proteasome in nanofluidic channels -

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Basic information

Entry
Database: EMDB / ID: EMD-12917
TitleElectron cryo-tomogram of T20S proteasome in nanofluidic channels
Map dataElectron cryo-tomogram
Sample
  • Complex: Thermoplasma acidophilium 20S proteasome
Biological speciesThermoplasma acidophilum (acidophilic)
Methodelectron tomography / cryo EM
AuthorsHuber ST / Sarajlic E / Huijink R / Evers WH / Jakobi AJ
Funding supportEuropean Union, Netherlands, 2 items
OrganizationGrant numberCountry
European Research Council (ERC)852880European Union
Netherlands Organisation for Scientific Research (NWO)NWO.STU.018-2.007 Netherlands
Citation
Journal: Elife / Year: 2022
Title: Nanofluidic chips for cryo-EM structure determination from picoliter sample volumes.
Authors: Stefan T Huber / Edin Sarajlic / Roeland Huijink / Felix Weis / Wiel H Evers / Arjen J Jakobi /
Abstract: Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness ...Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness still represents a barrier for routine high-resolution imaging and limits the current throughput of the technique. We show that a nanofluidic sample support with well-defined geometry can be used to prepare cryo-EM specimens with reproducible ice thickness from picoliter sample volumes. The sample solution is contained in electron-transparent nanochannels that provide uniform thickness gradients without further optimisation and eliminate the potentially destructive air-water interface. We demonstrate the possibility to perform high-resolution structure determination with three standard protein specimens. Nanofabricated sample supports bear potential to automate the cryo-EM workflow, and to explore new frontiers for cryo-EM applications such as time-resolved imaging and high-throughput screening.
#1: Journal: Biorxiv / Year: 2021
Title: Nanofluidic chips for cryo-EM structure determination from picoliter sample volumes
Authors: Huber ST / Sarajlic E / Huijink R / Weis F / Evers WH / Jakobi AJ
History
DepositionMay 11, 2021-
Header (metadata) releaseAug 11, 2021-
Map releaseAug 11, 2021-
UpdateFeb 16, 2022-
Current statusFeb 16, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
  • Download
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Movie viewer
Supplemental images

emd_12917.png

Downloads & links

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Map

FileDownload / File: emd_12917.map.gz / Format: CCP4 / Size: 3.2 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationElectron cryo-tomogram
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
7.34 Å/pix.
x 240 pix.
= 1760.64 Å		7.34 Å/pix.
x 1920 pix.
= 14085.12 Å		7.34 Å/pix.
x 1890 pix.
= 13865.04 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 7.336 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-3.0 - 3.0
Average (Standard dev.)-0.013128392 (±0.7874679)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions19201890240
Spacing18901920240
CellA: 13865.04 Å / B: 14085.12 Å / C: 1760.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z7.3367.3367.336
M x/y/z18901920240
origin x/y/z0.0000.0000.000
length x/y/z13865.04014085.1201760.640
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS18901920240
D min/max/mean-3.0003.000-0.013

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Supplemental data

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Additional map: Segmentation of T20S proteasomes

Fileemd_12917_additional_1.map
AnnotationSegmentation of T20S proteasomes
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Segmentation of the SiN membrane

Fileemd_12917_additional_2.map
AnnotationSegmentation of the SiN membrane
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Thermoplasma acidophilium 20S proteasome

EntireName: Thermoplasma acidophilium 20S proteasome
Components
  • Complex: Thermoplasma acidophilium 20S proteasome

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Supramolecule #1: Thermoplasma acidophilium 20S proteasome

SupramoleculeName: Thermoplasma acidophilium 20S proteasome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Thermoplasma acidophilum (acidophilic)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 700 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

Concentration1.4 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMNaClsodium chloride
20.0 mMTristris(hydroxymethyl)aminomethane
0.1 mMEDTAethylenediaminetetraacetic acid
GridModel: Homemade / Material: SILICON NITRIDE
VitrificationCryogen name: ETHANE / Instrument: LEICA PLUNGER
Details: The sample was filled into cryoChips through the cantilever and then transferred within ~10 seconds to the Leica plunger for freezing..
DetailsThe sample was filled into nanofluidic channels.
SectioningOther: NO SECTIONING

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Electron microscopy

MicroscopeJEOL 3200FSC
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 94.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 4.1 mm
Sample stageSpecimen holder model: JEOL 3200FSC CRYOHOLDER

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD (ver. 4.9.2) / Number images used: 121
CTF correctionSoftware - Name: IMOD (ver. 4.9.2)

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