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- EMDB-50110: Electron tomogram of ER-nuclear envelope junction of HeLa cell in... -

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Basic information

Entry
Database: EMDB / ID: EMD-50110
TitleElectron tomogram of ER-nuclear envelope junction of HeLa cell in early telophase
Map data
Sample
  • Cell: HeLa Kyoto cell
KeywordsEndoplasmic reticulum / Nuclear envelope / Organelle contact site / Membrane junction / CELL CYCLE
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM / negative staining
AuthorsBragulat-Teixidor H / Otsuka S
Funding support Austria, 2 items
OrganizationGrant numberCountry
Vienna Science and Technology Fund (WWTF)LS19-001 Austria
Austrian Science FundP 36743-B Austria
CitationJournal: EMBO Rep / Year: 2024
Title: The endoplasmic reticulum connects to the nucleus by constricted junctions that mature after mitosis.
Authors: Helena Bragulat-Teixidor / Keisuke Ishihara / Gréta Martina Szücs / Shotaro Otsuka /
Abstract: Junctions between the endoplasmic reticulum (ER) and the outer membrane of the nuclear envelope (NE) physically connect both organelles. These ER-NE junctions are essential for supplying the NE with ...Junctions between the endoplasmic reticulum (ER) and the outer membrane of the nuclear envelope (NE) physically connect both organelles. These ER-NE junctions are essential for supplying the NE with lipids and proteins synthesized in the ER. However, little is known about the structure of these ER-NE junctions. Here, we systematically study the ultrastructure of ER-NE junctions in cryo-fixed mammalian cells staged in anaphase, telophase, and interphase by correlating live cell imaging with three-dimensional electron microscopy. Our results show that ER-NE junctions in interphase cells have a pronounced hourglass shape with a constricted neck of 7-20 nm width. This morphology is significantly distinct from that of junctions within the ER network, and their morphology emerges as early as telophase. The highly constricted ER-NE junctions are seen in several mammalian cell types, but not in budding yeast. We speculate that the unique and highly constricted ER-NE junctions are regulated via novel mechanisms that contribute to ER-to-NE lipid and protein traffic in higher eukaryotes.
History
DepositionApr 16, 2024-
Header (metadata) releaseJun 19, 2024-
Map releaseJun 19, 2024-
UpdateJun 26, 2024-
Current statusJun 26, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50110.png

Downloads & links

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Map

FileDownload / File: emd_50110.map.gz / Format: CCP4 / Size: 20.6 GB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.51 Å/pix.
x 660 pix.
= 2976.6 Å		4.51 Å/pix.
x 4096 pix.
= 18472.961 Å		4.51 Å/pix.
x 4096 pix.
= 18472.961 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: 4.51 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-1808.0 - 1291.0
Average (Standard dev.)211.203519999999997 (±152.433690000000013)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-310
Dimensions40964096660
Spacing40964096660
CellA: 18472.96 Å / B: 18472.96 Å / C: 2976.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : HeLa Kyoto cell

EntireName: HeLa Kyoto cell
Components
  • Cell: HeLa Kyoto cell

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Supramolecule #1: HeLa Kyoto cell

SupramoleculeName: HeLa Kyoto cell / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human) / Strain: HeLa

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.4
Details: DMEM without Riboflavin and Phenol Red, containing 10% FBS, 1% Pen/Strep, and 50 nM SiR-DNA
StainingType: NEGATIVE / Material: Uranyl acetate and lead citrate
Sugar embeddingMaterial: Agar 100 Epoxy resin
Details: Frozen cells were substituted in 0.1% uranyl acetate (UA), 2% Osmium tetroxide and 5% H2O in acetone following this temperature ramp: -90 C to -80 C for 10 hours, -80 C to -30 C for 10 ...Details: Frozen cells were substituted in 0.1% uranyl acetate (UA), 2% Osmium tetroxide and 5% H2O in acetone following this temperature ramp: -90 C to -80 C for 10 hours, -80 C to -30 C for 10 hours, -30 C for 4 hours, -30 C to 0 C for 6 ours, 0 C to 20 C for 4 hours, 20 C for 5-6 hours.
VitrificationCryogen name: NITROGEN
High pressure freezingInstrument: OTHER
Details: High pressure freezing chamber was 1 mm thick, 6.0 mm diameter, with central cavities 5.0 mm x 5.0 mm x 25 um deep. The chamber had been in contact with 1-hexadecene.. The value given for _ ...Details: High pressure freezing chamber was 1 mm thick, 6.0 mm diameter, with central cavities 5.0 mm x 5.0 mm x 25 um deep. The chamber had been in contact with 1-hexadecene.. The value given for _em_high_pressure_freezing.instrument is Leica EM ICE. This is not in a list of allowed values {'LEICA EM PACT', 'LEICA EM PACT2', 'BAL-TEC HPM 010', 'OTHER', 'LEICA EM HPM100', 'EMS-002 RAPID IMMERSION FREEZER'} so OTHER is written into the XML file.
Cryo protectant20% Ficoll-PM400
SectioningUltramicrotomy - Instrument: Leica Ultracut UCT / Ultramicrotomy - Temperature: 298 K / Ultramicrotomy - Final thickness: 250
Fiducial markerManufacturer: Cytodiagnostics / Diameter: 15 nm

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: FEI EAGLE (4k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.2 µm
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Number images used: 110

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