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- EMDB-12902: Electron cryo-tomogram of pyrococcus furiosus apoferritin in nano... -

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Basic information

Entry
Database: EMDB / ID: EMD-12902
TitleElectron cryo-tomogram of pyrococcus furiosus apoferritin in nanofluidic channels
Map dataTomogram of nanofluidic channels filled with 3.4 mg/mL apoferritin solution
Sample
  • Complex: Nanochannel filled with pyrococcus furiosus apoferritin
Biological speciesPyrococcus furiosus COM1 (archaea)
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 10, 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_12902.png

Downloads & links

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Map

FileDownload / File: emd_12902.map.gz / Format: CCP4 / Size: 1.2 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTomogram of nanofluidic channels filled with 3.4 mg/mL apoferritin solution
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
9.41 Å/pix.
x 168 pix.
= 1581.216 Å		9.41 Å/pix.
x 1152 pix.
= 10842.624 Å		9.41 Å/pix.
x 1728 pix.
= 16263.936 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: 9.412 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-3.0 - 3.0
Average (Standard dev.)-0.011853187 (±0.73778814)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions11521728168
Spacing17281152168
CellA: 16263.936 Å / B: 10842.624 Å / C: 1581.216 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z9.4129.4129.412
M x/y/z17281152168
origin x/y/z0.0000.0000.000
length x/y/z16263.93610842.6241581.216
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS17281152168
D min/max/mean-3.0003.000-0.012

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Supplemental data

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Additional map: Segmentation of apoferritin particles

Fileemd_12902_additional_1.map
AnnotationSegmentation of apoferritin particles
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Segmentation of the silicon nitride membrane

Fileemd_12902_additional_2.map
AnnotationSegmentation of the silicon nitride 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 : Nanochannel filled with pyrococcus furiosus apoferritin

EntireName: Nanochannel filled with pyrococcus furiosus apoferritin
Components
  • Complex: Nanochannel filled with pyrococcus furiosus apoferritin

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Supramolecule #1: Nanochannel filled with pyrococcus furiosus apoferritin

SupramoleculeName: Nanochannel filled with pyrococcus furiosus apoferritin
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Pyrococcus furiosus COM1 (archaea)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 492 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

Concentration3.4 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMNaClsodium chloride
20.0 mMTrisris(hydroxymethyl)aminomethane
GridModel: Homemade / Material: SILICON NITRIDE
VitrificationCryogen name: ETHANE / Instrument: LEICA PLUNGER
Details: cryoChips were filled with 3.4 mg/mL apoferritin and transferred to the Leica plunger within ~10 seconds for plunging..
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 / Nominal defocus min: 6.0 µm

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Resolution method: OTHER / Number images used: 61
CTF correctionSoftware - Name: IMOD (ver. 4.9.2)

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