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- EMDB-15060: Fibroblast nucleus sub-volume. -

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Basic information

Entry
Database: EMDB / ID: EMD-15060
TitleFibroblast nucleus sub-volume.
Map datasub-volume of EMD-14924. corresponds to Supplementary Movie S5.
Sample
  • Cell: WI-38 fibroblast cell
Keywordsnucleus / chromatin / STEM / DNA
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsSedat J / McDonald A / Elbaum M
Funding support Israel, 1 items
OrganizationGrant numberCountry
Israel Science Foundation1696/18 Israel
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: A proposed unified interphase nucleus chromosome structure: Preliminary preponderance of evidence.
Authors: John Sedat / Angus McDonald / Hu Cang / Joseph Lucas / Muthuvel Arigovindan / Zvi Kam / Cornelis Murre / Michael Elbaum /
Abstract: Cryoelectron tomography of the cell nucleus using scanning transmission electron microscopy and deconvolution processing technology has highlighted a large-scale, 100- to 300-nm interphase chromosome ...Cryoelectron tomography of the cell nucleus using scanning transmission electron microscopy and deconvolution processing technology has highlighted a large-scale, 100- to 300-nm interphase chromosome structure, which is present throughout the nucleus. This study further documents and analyzes these chromosome structures. The paper is divided into four parts: 1) evidence (preliminary) for a unified interphase chromosome structure; 2) a proposed unified interphase chromosome architecture; 3) organization as chromosome territories (e.g., fitting the 46 human chromosomes into a 10-μm-diameter nucleus); and 4) structure unification into a polytene chromosome architecture and lampbrush chromosomes. Finally, the paper concludes with a living light microscopy cell study showing that the G1 nucleus contains very similar structures throughout. The main finding is that this chromosome structure appears to coil the 11-nm nucleosome fiber into a defined hollow structure, analogous to a Slinky helical spring [https://en.wikipedia.org/wiki/Slinky; motif used in Bowerman , 10, e65587 (2021)]. This Slinky architecture can be used to build chromosome territories, extended to the polytene chromosome structure, as well as to the structure of lampbrush chromosomes.
History
DepositionMay 30, 2022-
Header (metadata) releaseJul 6, 2022-
Map releaseJul 6, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15060.png

Downloads & links

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Map

FileDownload / File: emd_15060.map.gz / Format: CCP4 / Size: 63.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsub-volume of EMD-14924. corresponds to Supplementary Movie S5.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
33.5 Å/pix.
x 100 pix.
= 3350. Å		33.5 Å/pix.
x 470 pix.
= 15745. Å		33.5 Å/pix.
x 356 pix.
= 11926. 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: 33.5 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum1500.0 - 8000.0
Average (Standard dev.)1673.895999999999958 (±151.96153000000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin18040273
Dimensions470356100
Spacing356470100
CellA: 11926.0 Å / B: 15745.0 Å / C: 3350.0 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.
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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 20.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 0.0 µm / Nominal defocus min: 0.0 µm
Specialist opticsDetails: STEM data collection in BF mode.
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
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
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

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
DetailsGatan STEM BF/ADF detector

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