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Yorodumi- EMDB-19161: Thinner is not always better: Optimising cryo lamellae for subtom... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-19161 | |||||||||
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Title | Thinner is not always better: Optimising cryo lamellae for subtomogram averaging - Ion-damage layer analysis | |||||||||
Map data | In-situ ribosome from Dictyostelium discoideum obtained with subtomogram averaging. Ion-damage analysis: 50 nm from lamella surface | |||||||||
Sample |
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Keywords | In-situ Dictyostelium discoideum ribosome / RIBOSOME | |||||||||
Biological species | Dictyostelium discoideum AX2 (eukaryote) | |||||||||
Method | subtomogram averaging / cryo EM / Resolution: 13.7 Å | |||||||||
Authors | Tuijtel MW / Cruz-Leon S / Kreysing JP / Welsch S / Hummer G / Beck M / Turonova B | |||||||||
Funding support | United States, Germany, 2 items
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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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_19161.map.gz / Format: CCP4 / Size: 18.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | In-situ ribosome from Dictyostelium discoideum obtained with subtomogram averaging. Ion-damage analysis: 50 nm from lamella surface | ||||||||||||||||||||
Voxel size | X=Y=Z: 2.446 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Additional map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Half map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
+Half map: In-situ ribosome from Dictyostelium discoideum obtained with subtomogram...
-Sample components
-Entire : Ribosome
Entire | Name: Ribosome |
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Components |
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-Supramolecule #1: Ribosome
Supramolecule | Name: Ribosome / type: complex / ID: 1 / Parent: 0 / Details: Ribosome |
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Source (natural) | Organism: Dictyostelium discoideum AX2 (eukaryote) / Location in cell: cytoplasm |
Molecular weight | Theoretical: 4 MDa |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | subtomogram averaging |
Aggregation state | cell |
-Sample preparation
Buffer | pH: 6.5 |
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Vitrification | Cryogen name: ETHANE / Chamber humidity: 70 % / Chamber temperature: 20 K / Instrument: LEICA EM GP |
-Electron microscopy
Microscope | TFS KRIOS |
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Specialist optics | Energy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV |
Image recording | Film 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 beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 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 stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |