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- EMDB-19671: In situ cryo-electron tomogram of Saccharomyces cerevisiae -

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Basic information

Entry
Database: EMDB / ID: EMD-19671
TitleIn situ cryo-electron tomogram of Saccharomyces cerevisiae
Map data
Sample
  • Cell: Saccharomyces cerevisiae (yeast)
KeywordsSaccharomyces cerevisiae / yeast / UNKNOWN FUNCTION
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodelectron tomography / cryo EM
AuthorsComet M / Dijkman PM / Boer Iwema R / Franke T / Masiulis S / Schampers R / Raschdorf O / Grollios F / Pryor Jr EE / Drulyte I
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2024
Title: Tomo Live: an on-the-fly reconstruction pipeline to judge data quality for cryo-electron tomography workflows.
Authors: Maxime Comet / Patricia M Dijkman / Reint Boer Iwema / Tilman Franke / Simonas Masiulis / Ruud Schampers / Oliver Raschdorf / Fanis Grollios / Edward E Pryor / Ieva Drulyte /
Abstract: Data acquisition and processing for cryo-electron tomography can be a significant bottleneck for users. To simplify and streamline the cryo-ET workflow, Tomo Live, an on-the-fly solution that ...Data acquisition and processing for cryo-electron tomography can be a significant bottleneck for users. To simplify and streamline the cryo-ET workflow, Tomo Live, an on-the-fly solution that automates the alignment and reconstruction of tilt-series data, enabling real-time data-quality assessment, has been developed. Through the integration of Tomo Live into the data-acquisition workflow for cryo-ET, motion correction is performed directly after each of the acquired tilt angles. Immediately after the tilt-series acquisition has completed, an unattended tilt-series alignment and reconstruction into a 3D volume is performed. The results are displayed in real time in a dedicated remote web platform that runs on the microscope hardware. Through this web platform, users can review the acquired data (aligned stack and 3D volume) and several quality metrics that are obtained during the alignment and reconstruction process. These quality metrics can be used for fast feedback for subsequent acquisitions to save time. Parameters such as Alignment Accuracy, Deleted Tilts and Tilt Axis Correction Angle are visualized as graphs and can be used as filters to export only the best tomograms (raw data, reconstruction and intermediate data) for further processing. Here, the Tomo Live algorithms and workflow are described and representative results on several biological samples are presented. The Tomo Live workflow is accessible to both expert and non-expert users, making it a valuable tool for the continued advancement of structural biology, cell biology and histology.
History
DepositionFeb 17, 2024-
Header (metadata) releaseFeb 28, 2024-
Map releaseFeb 28, 2024-
UpdateApr 17, 2024-
Current statusApr 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19671.png

Downloads & links

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Map

FileDownload / File: emd_19671.map.gz / Format: CCP4 / Size: 616 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
7.9 Å/pix.
x 308 pix.
= 2433.2 Å		7.9 Å/pix.
x 1024 pix.
= 8089.6 Å		7.9 Å/pix.
x 1024 pix.
= 8089.6 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: 7.9 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-32768.0 - 32767.0
Average (Standard dev.)4257.669399999999769 (±4541.545399999999972)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions10241024308
Spacing10241024308
CellA: 8089.6 Å / B: 8089.6 Å / C: 2433.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Saccharomyces cerevisiae (yeast)

EntireName: Saccharomyces cerevisiae (yeast)
Components
  • Cell: Saccharomyces cerevisiae (yeast)

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Supramolecule #1: Saccharomyces cerevisiae (yeast)

SupramoleculeName: Saccharomyces cerevisiae (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

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7
VitrificationCryogen name: ETHANE
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 0.5 / Focused ion beam - Duration: 2400 / Focused ion beam - Temperature: 91 K / Focused ion beam - Initial thickness: 7000 / Focused ion beam - Final thickness: 150
Focused ion beam - Details: Rough milling with 300-1000 pA, polishing with 10-100 pA.. The value given for _em_focused_ion_beam.instrument is TFS Aquilos 2. This is not in a list of allowed values ...Focused ion beam - Details: Rough milling with 300-1000 pA, polishing with 10-100 pA.. The value given for _em_focused_ion_beam.instrument is TFS Aquilos 2. This is not in a list of allowed values {'OTHER', 'DB235'} so OTHER is written into the XML file.

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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) / Number real images: 37 / Average electron dose: 3.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.0 µm / Nominal defocus min: 4.0 µm / Nominal magnification: 64000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: SIMULTANEOUS ITERATIVE (SIRT)
Details: Reconstruction was obtained using Tomo Live on-the-fly reconstruction pipeline
Number images used: 37

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