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- EMDB-3820: Electron tomographic slices of the nuclear envelope of HeLa cell ... -

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Basic information

Entry
Database: EMDB / ID: EMD-3820
TitleElectron tomographic slices of the nuclear envelope of HeLa cell in interphase
Map dataNone
Sample
  • Cell: HeLa cellHeLa
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM / negative staining
AuthorsOtsuka S / Ellenberg J
CitationJournal: Nat Struct Mol Biol / Year: 2018
Title: Postmitotic nuclear pore assembly proceeds by radial dilation of small membrane openings.
Authors: Shotaro Otsuka / Anna M Steyer / Martin Schorb / Jean-Karim Hériché / M Julius Hossain / Suruchi Sethi / Moritz Kueblbeck / Yannick Schwab / Martin Beck / Jan Ellenberg /
Abstract: The nuclear envelope has to be reformed after mitosis to create viable daughter cells with closed nuclei. How membrane sealing of DNA and assembly of nuclear pore complexes (NPCs) are achieved and ...The nuclear envelope has to be reformed after mitosis to create viable daughter cells with closed nuclei. How membrane sealing of DNA and assembly of nuclear pore complexes (NPCs) are achieved and coordinated is poorly understood. Here, we reconstructed nuclear membrane topology and the structures of assembling NPCs in a correlative 3D EM time course of dividing human cells. Our quantitative ultrastructural analysis shows that nuclear membranes form from highly fenestrated ER sheets whose holes progressively shrink. NPC precursors are found in small membrane holes and dilate radially during assembly of the inner ring complex, forming thousands of transport channels within minutes. This mechanism is fundamentally different from that of interphase NPC assembly and explains how mitotic cells can rapidly establish a closed nuclear compartment while making it transport competent.
History
DepositionJul 20, 2017-
Header (metadata) releaseAug 23, 2017-
Map releaseNov 29, 2017-
UpdateJan 24, 2018-
Current statusJan 24, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Simplified surface model
  • Imaged by Jmol
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3820.png

Downloads & links

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Map

FileDownload / File: emd_3820.map.gz / Format: CCP4 / Size: 1.8 GB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 7.5 Å
Density
Minimum - Maximum-4205. - 1647.
Average (Standard dev.)173.766950000000008 (±219.101499999999987)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00150
Dimensions40964096326
Spacing40964096326
CellA: 30720.0 Å / B: 30720.0 Å / C: 2445.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z7.57.57.5
M x/y/z40964096326
origin x/y/z0.0000.0000.000
length x/y/z30720.00030720.0002445.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS00150
NC/NR/NS40964096326
D min/max/mean-4205.0001647.000173.767

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Supplemental data

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Sample components

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Entire : HeLa cell

EntireName: HeLa cellHeLa
Components
  • Cell: HeLa cellHeLa

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Supramolecule #1: HeLa cell

SupramoleculeName: HeLa cell / type: cell / ID: 1 / Parent: 0
Details: Cells were high-pressure frozen and freeze-substituted into Lowicryl resin.
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: CO2-independent medium without phenol red (Invitrogen), containing 20% FCS, 20% Ficoll PM400 2 mM l-glutamine, and 100 ug/ml penicillin and streptomycin
StainingType: NEGATIVE / Material: uranyl acetate and lead citrate
Sugar embeddingMaterial: Lowicryl resin
Details: Frozen cells were incubated with 0.1% uranyl acetate in acetone at -90C for 20-24 hr and, after infiltration into Lowicryl resin and UV-polymerization, samples were further polymerized by sunlight for 3-4 days.
GridModel: Grid / Support film - Material: FORMVAR / Support film - topology: CONTINUOUS
VitrificationCryogen name: NITROGEN
High pressure freezingInstrument: OTHER
Details: High pressure freezing chamber was 1.0 mm thick in total, 3.0 mm diameter, with central cavities 50 um deep.. The value given for _emd_high_pressure_freezing.instrument is HPM 010. This is ...Details: High pressure freezing chamber was 1.0 mm thick in total, 3.0 mm diameter, with central cavities 50 um deep.. The value given for _emd_high_pressure_freezing.instrument is HPM 010. This is not in a list of allowed values set(['LEICA EM PACT2', 'LEICA EM PACT', 'EMS-002 RAPID IMMERSION FREEZER', 'OTHER', 'LEICA EM HPM100', 'BAL-TEC HPM 010']) so OTHER is written into the XML file.
Cryo protectant20% FBS and Ficoll PM400
SectioningUltramicrotomy - Instrument: Leica Ultracut UCT / Ultramicrotomy - Temperature: 25 K / Ultramicrotomy - Final thickness: 300
Fiducial markerManufacturer: CMC university Medical Center Utrecht / Diameter: 15 nm

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus min: 0.5 µm / Nominal magnification: 15500
Sample stageSpecimen holder model: OTHER
Image recordingFilm or detector model: FEI EAGLE (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number real images: 121 / Average electron dose: 200.0 e/Å2
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: IMOD (ver. 4.5.6)
Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD (ver. 4.5.6)
Details: Dual axis tilt series were aligned using gold fiducial markers
Number images used: 121

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