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- EMDB-19136: Thinner is not always better: Optimising cryo lamellae for subtom... -

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Entry
Database: EMDB / ID: EMD-19136
TitleThinner is not always better: Optimising cryo lamellae for subtomogram averaging
Map dataRibosome from Dictyostelium discoideum, from in-situ data using cryo-ET and STA. "high-quality" particles, 23k particles.
Sample
  • Complex: Ribosome
KeywordsIn-situ Dictyostelium discoideum ribosome / RIBOSOME
Biological speciesDictyostelium discoideum AX2 (eukaryote)
Methodsubtomogram averaging / cryo EM / Resolution: 3.9 Å
AuthorsTuijtel MW / Cruz-Leon S / Kreysing JP / Welsch S / Hummer G / Beck M / Turonova B
Funding support United States, Germany, 2 items
OrganizationGrant numberCountry
Chan Zuckerberg Initiative2021-234666 United States
Max Planck Society Germany
Citation
Journal: Sci Adv / Year: 2024
Title: Thinner is not always better: Optimizing cryo-lamellae for subtomogram averaging.
Authors: Maarten W Tuijtel / Sergio Cruz-León / Jan Philipp Kreysing / Sonja Welsch / Gerhard Hummer / Martin Beck / Beata Turoňová /
Abstract: Cryo-electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and to structurally analyze biomolecules in situ by subtomogram averaging, yet data quality critically ...Cryo-electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and to structurally analyze biomolecules in situ by subtomogram averaging, yet data quality critically depends on specimen thickness. Cells that are too thick for transmission imaging can be thinned into lamellae by cryo-focused ion beam (cryo-FIB) milling. Despite being a crucial parameter directly affecting attainable resolution, optimal lamella thickness has not been systematically investigated nor the extent of structural damage caused by gallium ions used for FIB milling. We thus systematically determined how resolution is affected by these parameters. We find that ion-induced damage does not affect regions more than 30 nanometers from either lamella surface and that up to ~180-nanometer lamella thickness does not negatively affect resolution. This shows that there is no need to generate very thin lamellae and lamella thickness can be chosen such that it captures cellular features of interest, thereby opening cryo-ET also for studies of large complexes.
#1: Journal: BiorXiv
Title: Thinner is not always better: Optimising cryo lamellae for subtomogram averaging
Authors: Tuijtel MW / Cruz-Leon S / Kreysing JP / Welsch S / Hummer G / Beck M / Turonova B
History
DepositionDec 14, 2023-
Header (metadata) releaseMay 15, 2024-
Map releaseMay 15, 2024-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19136.png

Downloads & links

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Map

FileDownload / File: emd_19136.map.gz / Format: CCP4 / Size: 87.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRibosome from Dictyostelium discoideum, from in-situ data using cryo-ET and STA. "high-quality" particles, 23k particles.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.22 Å/pix.
x 284 pix.
= 347.332 Å		1.22 Å/pix.
x 284 pix.
= 347.332 Å		1.22 Å/pix.
x 284 pix.
= 347.332 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.223 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00196
Minimum - Maximum-0.005388909 - 0.009880301
Average (Standard dev.)0.00008011366 (±0.00075962354)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions284284284
Spacing284284284
CellA=B=C: 347.332 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Ribosome from Dictyostelium discoideum, from in-situ data using...

Fileemd_19136_additional_1.map
AnnotationRibosome from Dictyostelium discoideum, from in-situ data using cryo-ET and STA. "random" particles, 23k particles.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Ribosome from Dictyostelium discoideum, from in-situ data using...

Fileemd_19136_additional_2.map
AnnotationRibosome from Dictyostelium discoideum, from in-situ data using cryo-ET and STA. "random" particles, 23k particles. halfmap 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Ribosome from Dictyostelium discoideum, from in-situ data using...

Fileemd_19136_additional_3.map
AnnotationRibosome from Dictyostelium discoideum, from in-situ data using cryo-ET and STA. "random" particles, 23k particles. halfmap 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Ribosome from Dictyostelium discoideum, from in-situ data using...

Fileemd_19136_additional_4.map
AnnotationRibosome from Dictyostelium discoideum, from in-situ data using cryo-ET and STA. "high-quality" particles, 5k particles. Halfmap 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Ribosome from Dictyostelium discoideum, from in-situ data using...

Fileemd_19136_additional_5.map
AnnotationRibosome from Dictyostelium discoideum, from in-situ data using cryo-ET and STA. "high-quality" particles, 5k particles. Halfmap 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

+
Additional map: Ribosome from Dictyostelium discoideum, from in-situ data using...

Fileemd_19136_additional_6.map
AnnotationRibosome from Dictyostelium discoideum, from in-situ data using cryo-ET and STA. "high-quality" particles, 5k particles.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

+
Additional map: Ribosome from Dictyostelium discoideum, from in-situ data using...

Fileemd_19136_additional_7.map
AnnotationRibosome from Dictyostelium discoideum, from in-situ data using cryo-ET and STA. "random" particles, 5k particles. Halfmap 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

+
Additional map: Ribosome from Dictyostelium discoideum, from in-situ data using...

Fileemd_19136_additional_8.map
AnnotationRibosome from Dictyostelium discoideum, from in-situ data using cryo-ET and STA. "random" particles, 5k particles. Halfmap 2.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

+
Additional map: Ribosome from Dictyostelium discoideum, from in-situ data using...

Fileemd_19136_additional_9.map
AnnotationRibosome from Dictyostelium discoideum, from in-situ data using cryo-ET and STA. "random" particles, 5k particles.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap 1 "high-quality" particles, 23k particles.

Fileemd_19136_half_map_1.map
Annotationhalfmap 1 "high-quality" particles, 23k particles.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap 2 "high-quality" particles, 23k particles.

Fileemd_19136_half_map_2.map
Annotationhalfmap 2 "high-quality" particles, 23k particles.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ribosome

EntireName: Ribosome
Components
  • Complex: Ribosome

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Supramolecule #1: Ribosome

SupramoleculeName: Ribosome / type: complex / ID: 1 / Parent: 0 / Details: Ribosome
Source (natural)Organism: Dictyostelium discoideum AX2 (eukaryote) / Location in cell: cytoplasm
Molecular weightTheoretical: 4 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: 6.5
GridModel: Quantifoil / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Chamber temperature: 20 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number real images: 2 / Average exposure time: 0.24 sec. / Average electron dose: 2.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 2.5 µm
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 reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Warp / Number subtomograms used: 22586
ExtractionNumber tomograms: 261 / Number images used: 168000 / Method: Template matching / Software - Name: Warp
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: Warp
FSC plot (resolution estimation)

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