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- EMDB-42879: 3x3 tiled montage tomogram of a yeast lamella imaged with a squar... -

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Basic information

Entry
Database: EMDB / ID: EMD-42879
Title3x3 tiled montage tomogram of a yeast lamella imaged with a square electron beam
Map data
Sample
  • Cell: Yeast
KeywordsFIB-milled yeast / UNKNOWN FUNCTION
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodelectron tomography
AuthorsChua EYD / Alink LM / Kopylov M / Johnston J / Einsenstein F / de Marco A
Funding support United States, 2 items
OrganizationGrant numberCountry
Simons FoundationSF349247 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
CitationJournal: Nat Methods / Year: 2024
Title: Square beams for optimal tiling in transmission electron microscopy.
Authors: Eugene Y D Chua / Lambertus M Alink / Mykhailo Kopylov / Jake D Johnston / Fabian Eisenstein / Alex de Marco /
Abstract: Imaging large fields of view at a high magnification requires tiling. Transmission electron microscopes typically have round beam profiles; therefore, tiling across a large area is either imperfect ...Imaging large fields of view at a high magnification requires tiling. Transmission electron microscopes typically have round beam profiles; therefore, tiling across a large area is either imperfect or results in uneven exposures, a problem for dose-sensitive samples. Here, we introduce a square electron beam that can easily be retrofitted in existing microscopes, and demonstrate its application, showing that it can tile nearly perfectly and deliver cryo-electron microscopy imaging with a resolution comparable to conventional set-ups.
History
DepositionNov 19, 2023-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateApr 24, 2024-
Current statusApr 24, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42879.png

Downloads & links

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Map

FileDownload / File: emd_42879.map.gz / Format: CCP4 / Size: 13.7 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 8 Å
Density
Minimum - Maximum-1367.117999999999938 - 1220.756800000000112
Average (Standard dev.)-0.18173476 (±61.159480000000002)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin0-153-993
Dimensions153615471546
Spacing154715361546
CellA: 12376.0 Å / B: 12288.0 Å / C: 12368.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Yeast

EntireName: Yeast (yeast)
Components
  • Cell: Yeast

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Supramolecule #1: Yeast

SupramoleculeName: Yeast / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Experimental details

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Structure determination

Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.5 / Details: YPD media
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER / Details: Gatan Solarus I
DetailsExtrapolated OD 600 ~20
High pressure freezingInstrument: OTHER
Details: Sample was high-pressure frozen directly on a 200 mesh grid sandwiched between two flat sides of 3 mm planchettes. The value given for _em_high_pressure_freezing.instrument is Wohlwend HPF ...Details: Sample was high-pressure frozen directly on a 200 mesh grid sandwiched between two flat sides of 3 mm planchettes. The value given for _em_high_pressure_freezing.instrument is Wohlwend HPF Compact 01. This is not in a list of allowed values {'LEICA EM HPM100', 'OTHER', 'LEICA EM PACT', 'BAL-TEC HPM 010', 'EMS-002 RAPID IMMERSION FREEZER', 'LEICA EM PACT2'} so OTHER is written into the XML file.
Cryo protectant5% glycerol
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 2 / Focused ion beam - Duration: 300 / Focused ion beam - Temperature: 77 K / Focused ion beam - Initial thickness: 500 / Focused ion beam - Final thickness: 200
Focused ion beam - Details: Lamellae were prepared using TFS AutoTEM. The settings specified are for the final polishing only. The complete set of parameters is described in the Waffle Method ...Focused ion beam - Details: Lamellae were prepared using TFS AutoTEM. The settings specified are for the final polishing only. The complete set of parameters is described in the Waffle Method protocol paper here: https://ncbi.nlm.nih.gov/pmc/articles/PMC9795037/. The value given for _em_focused_ion_beam.instrument is Aquilos 2. This is not in a list of allowed values {'DB235', 'OTHER'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.5 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 2.55 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware - Name: TOMO3D / Number images used: 279

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