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- EMDB-14924: Cryo-STEM tomography of fibroblast nuclear periphery -

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Basic information

Entry
Database: EMDB / ID: EMD-14924
TitleCryo-STEM tomography of fibroblast nuclear periphery
Map dataCryo-STEM tomography (CSTET) of an intact fibroblast showing nuclear periphery, processed by 3D deconvolution.
Sample
  • Cell: WI-38 fibroblast cell
Keywordsnucleus / chromatin / nuclear envelope / mitochondria / STEM / CSTET / DNA
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsWolf SG / Fass D / Elbaum M
Funding support Israel, 1 items
OrganizationGrant numberCountry
Israel Science Foundation1696/18 Israel
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2020
Title: Three-dimensional deconvolution processing for STEM cryotomography.
Authors: Barnali Waugh / Sharon G Wolf / Deborah Fass / Eric Branlund / Zvi Kam / John W Sedat / Michael Elbaum /
Abstract: The complex environment of biological cells and tissues has motivated development of three-dimensional (3D) imaging in both light and electron microscopies. To this end, one of the primary tools in ...The complex environment of biological cells and tissues has motivated development of three-dimensional (3D) imaging in both light and electron microscopies. To this end, one of the primary tools in fluorescence microscopy is that of computational deconvolution. Wide-field fluorescence images are often corrupted by haze due to out-of-focus light, i.e., to cross-talk between different object planes as represented in the 3D image. Using prior understanding of the image formation mechanism, it is possible to suppress the cross-talk and reassign the unfocused light to its proper source post facto. Electron tomography based on tilted projections also exhibits a cross-talk between distant planes due to the discrete angular sampling and limited tilt range. By use of a suitably synthesized 3D point spread function, we show here that deconvolution leads to similar improvements in volume data reconstructed from cryoscanning transmission electron tomography (CSTET), namely a dramatic in-plane noise reduction and improved representation of features in the axial dimension. Contrast enhancement is demonstrated first with colloidal gold particles and then in representative cryotomograms of intact cells. Deconvolution of CSTET data collected from the periphery of an intact nucleus revealed partially condensed, extended structures in interphase chromatin.
#1: Journal: To Be Published
Title: A Proposed Unified Interphase Nucleus Chromosome Structure: Preliminary Preponderance of Evidence
Authors: Sedat J / McDonald A / Cang H / Lucas J / Arigovindan M / Kam Z / Murre C / Elbaum M
History
DepositionMay 6, 2022-
Header (metadata) releaseJun 15, 2022-
Map releaseJun 15, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14924.png

Downloads & links

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Map

FileDownload / File: emd_14924.map.gz / Format: CCP4 / Size: 2 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-STEM tomography (CSTET) of an intact fibroblast showing nuclear periphery, processed by 3D deconvolution.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
33.51 Å/pix.
x 512 pix.
= 17157.119 Å		33.51 Å/pix.
x 1024 pix.
= 34314.238 Å		33.51 Å/pix.
x 1024 pix.
= 34314.238 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: 33.51 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-4476.86869999999999 - 1470985.5
Average (Standard dev.)1849.800899999999956 (±542.536560000000009)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions10241024512
Spacing10241024512
CellA: 34314.24 Å / B: 34314.24 Å / C: 17157.12 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : WI-38 fibroblast cell

EntireName: WI-38 fibroblast cell
Components
  • Cell: WI-38 fibroblast cell

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Supramolecule #1: WI-38 fibroblast cell

SupramoleculeName: WI-38 fibroblast cell / type: cell / ID: 1 / Parent: 0 / Details: cells cultured directly on gold EM grid
Source (natural)Organism: Homo sapiens (human) / Organ: lung / Tissue: cell culture

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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: 7.4
Details: Minimal Essential Medium (Gibco) supplemented with 15% fetal calf serum, L-glutamine, and penicillin/streptomycin.
GridModel: Quantifoil R3.5/1 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Instrument: LEICA EM GP
DetailsWI-38 embryonic lung fibroblast cells (obtained from Coriell Institute) were cultured directly on gold EM grids and imaged intact by cryo-STEM tomography (CSTET).
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: obtained from L Duchesne (https://doi.org/10.1021/la802876u )
Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TECNAI F20
Specialist opticsDetails: STEM data collection in BF mode.
DetailsNanoProbe STEM acquisition.
Image recordingFilm or detector model: OTHER / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Number grids imaged: 1 / Number real images: 61 / Average exposure time: 12.6 sec. / Average electron dose: 3.8 e/Å2 / Details: Cryo-STEM tilt series
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 20.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 0.0 µm / Nominal defocus min: 0.0 µm
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

DetailsGatan STEM BF/ADF detector
Final reconstructionAlgorithm: BACK PROJECTION / Software: (Name: TOMO3D, PRIISM/IVE)
Details: The original alignment was done using etomo (IMOD), and then optimized using tomoalign as a "thin" specimen. A new aligned stack (.ali) was produced using tomowarpalign (tomoalign) and ...Details: The original alignment was done using etomo (IMOD), and then optimized using tomoalign as a "thin" specimen. A new aligned stack (.ali) was produced using tomowarpalign (tomoalign) and reconstructed by SIRT using tomo3d. The reconstruction was then binned (by 2) and processed by 3D deconvolution as described in Waugh et al (doi:10.1073/pnas.2000700117) with smoothing parameter 0.1.
Number images used: 56

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