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- EMDB-19762: 4D_STEM cryo tomogram of t4 Phages produced by PCA2 -

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Basic information

Entry
Database: EMDB / ID: EMD-19762
Title4D_STEM cryo tomogram of t4 Phages produced by PCA2
Map data4D_STEM cryo tomogram of t4 Phages produced by PCA2
Sample
  • Virus: Escherichia phage T4 (virus)
    • Organelle or cellular component: Escherichia phage T4
Keywords4D-STEM cryo tomography / Cryo-ET / CSTET / T4-Phage / iDPC / UNKNOWN FUNCTION
Biological speciesEscherichia phage T4 (virus)
Methodelectron tomography / cryo EM
AuthorsSeifer S / Kirchweger P / Edel KM / Elbaum M
Funding support Austria, Israel, European Union, 4 items
OrganizationGrant numberCountry
Austrian Science FundJ4449-B Austria
Israel Science Foundation1696/18 Israel
European Union (EU)IMpaCT (grant no.857203)European Union
European Research Council (ERC)101055413European Union
CitationJournal: Microsc Microanal / Year: 2024
Title: Optimizing Contrast in Automated 4D STEM Cryotomography.
Authors: Shahar Seifer / Peter Kirchweger / Karlina Maria Edel / Michael Elbaum /
Abstract: 4D STEM is an emerging approach to electron microscopy. While it was developed principally for high-resolution studies in materials science, the possibility to collect the entire transmitted flux ...4D STEM is an emerging approach to electron microscopy. While it was developed principally for high-resolution studies in materials science, the possibility to collect the entire transmitted flux makes it attractive for cryomicroscopy in application to life science and radiation-sensitive materials where dose efficiency is of utmost importance. We present a workflow to acquire tomographic tilt series of 4D STEM data sets using a segmented diode and an ultrafast pixelated detector, demonstrating the methods using a specimen of a T4 bacteriophage. Full integration with the SerialEM platform conveniently provides all the tools for grid navigation and automation of the data collection. Scripts are provided to convert the raw data to mrc format files and further to generate a variety of modes representing both scattering and phase contrasts, including incoherent and annular bright field, integrated center of mass, and parallax decomposition of a simulated integrated differential phase contrast. Principal component analysis of virtual annular detectors proves particularly useful, and axial contrast is improved by 3D deconvolution with an optimized point spread function. Contrast optimization enables visualization of irregular features such as DNA strands and thin filaments of the phage tails, which would be lost upon averaging or imposition of an inappropriate symmetry.
History
DepositionFeb 29, 2024-
Header (metadata) releaseMay 8, 2024-
Map releaseMay 8, 2024-
UpdateJun 26, 2024-
Current statusJun 26, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19762.png

Downloads & links

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Map

FileDownload / File: emd_19762.map.gz / Format: CCP4 / Size: 964 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation4D_STEM cryo tomogram of t4 Phages produced by PCA2
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
15.58 Å/pix.
x 241 pix.
= 3754.78 Å		15.58 Å/pix.
x 1024 pix.
= 15953.92 Å		15.58 Å/pix.
x 1024 pix.
= 15953.92 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: 15.58 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-10582.227000000000771 - 22332.944999999999709
Average (Standard dev.)2200.836999999999989 (±2072.56860000000006)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-895102
Dimensions10241024241
Spacing10241024241
CellA: 15953.92 Å / B: 15953.92 Å / C: 3754.78 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Escherichia phage T4

EntireName: Escherichia phage T4 (virus)
Components
  • Virus: Escherichia phage T4 (virus)
    • Organelle or cellular component: Escherichia phage T4

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Supramolecule #1: Escherichia phage T4

SupramoleculeName: Escherichia phage T4 / type: virus / ID: 1 / Parent: 0 / Details: Isolated T4 Bacteriophage / NCBI-ID: 2681598 / Sci species name: Escherichia phage T4 / Virus type: VIROID / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No
Host (natural)Organism: Escherichia coli DH5[alpha] (bacteria) / Strain: DH5a

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Supramolecule #2: Escherichia phage T4

SupramoleculeName: Escherichia phage T4 / type: organelle_or_cellular_component / ID: 2 / Parent: 1
Source (natural)Organism: Escherichia phage T4 (virus)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4 / Details: 50 mM Tris pH 8 with 1 mM MgCl2
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #0 - topology: HOLEY / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: CARBON / Support film - #1 - topology: CONTINUOUS / Support film - #1 - Film thickness: 2
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 296.15 K / Instrument: LEICA EM GP
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Home made / Diameter: 15 nm

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Electron microscopy

MicroscopeFEI TECNAI F20
Details200 kV with 30 um C2 aperture, for a semi-convergence angle of 0.8 mrad and typically 7-10 pA electron current
Image recordingFilm or detector model: DECTRIS ARINA (0.2k x 0.2k) / Digitization - Dimensions - Width: 1024 pixel / Digitization - Dimensions - Height: 1024 pixel / Average electron dose: 3.0 e/Å2
Details: Images were recorded on a Dectris ARINA 4D-STEM detector.
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 30.0 µm / Calibrated defocus min: 0.0 µm / Illumination mode: SPOT SCAN / Imaging mode: 4D-STEM / Nominal defocus max: 0.0 µm / Nominal defocus min: 0.0 µm / Nominal magnification: 41000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsThe tilt-images were generated from 4D-STEM data using the iDPC2 algorithm (Seifer et.al., 2024).
Final reconstructionAlgorithm: BACK PROJECTION
Software:
Namedetails
MATLAB
PRIISM/IVE (ver. 4.7.2)Deconvolved using core2_decon

Number images used: 41

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