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- EMDB-6846: Volta phase contrast cryotomogram of a cryosectioned prometaphase... -

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Entry
Database: EMDB / ID: EMD-6846
TitleVolta phase contrast cryotomogram of a cryosectioned prometaphase S. pombe cell, strain nda3-KM311
Map dataVolta phase contrast cryotomogram of a cryosectioned prometaphase S. pombe cell, strain nda3-KM311
Sample
  • Cell: S. pombe cell, arrested in prometaphase
Biological speciesSchizosaccharomyces pombe (fission yeast)
Methodelectron tomography / cryo EM
AuthorsCai S / Chen C / Tan Z / Huang Y / Shi J / Gan L
Funding support Singapore, 5 items
OrganizationGrant numberCountry
NUSR-154-000-515-133 Singapore
NUSD-E12-303-154-217 Singapore
NUSR-154-000-524-651 Singapore
MOER-154-000-624-112 Singapore
NUSR-154-000-558-133 Singapore
CitationJournal: Proc Natl Acad Sci U S A / Year: 2018
Title: Cryo-ET reveals the macromolecular reorganization of mitotic chromosomes in vivo.
Authors: Shujun Cai / Chen Chen / Zhi Yang Tan / Yinyi Huang / Jian Shi / Lu Gan /
Abstract: Chromosomes condense during mitosis in most eukaryotes. This transformation involves rearrangements at the nucleosome level and has consequences for transcription. Here, we use cryo-electron ...Chromosomes condense during mitosis in most eukaryotes. This transformation involves rearrangements at the nucleosome level and has consequences for transcription. Here, we use cryo-electron tomography (cryo-ET) to determine the 3D arrangement of nuclear macromolecular complexes, including nucleosomes, in frozen-hydrated cells. Using 3D classification analysis, we did not find evidence that nucleosomes resembling the crystal structure are abundant. This observation and those from other groups support the notion that a subset of fission yeast nucleosomes may be partially unwrapped in vivo. In both interphase and mitotic cells, there is also no evidence of monolithic structures the size of Hi-C domains. The chromatin is mingled with two features: pockets, which are positions free of macromolecular complexes; and "megacomplexes," which are multimegadalton globular complexes like preribosomes. Mitotic chromatin is more crowded than interphase chromatin in subtle ways. Nearest-neighbor distance analyses show that mitotic chromatin is more compacted at the oligonucleosome than the dinucleosome level. Like interphase, mitotic chromosomes contain megacomplexes and pockets. This uneven chromosome condensation helps explain a longstanding enigma of mitosis: a subset of genes is up-regulated.
History
DepositionNov 21, 2017-
Header (metadata) releaseOct 31, 2018-
Map releaseOct 31, 2018-
UpdateAug 5, 2020-
Current statusAug 5, 2020Processing site: PDBj / 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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Supplemental images

emd_6846.png

Downloads & links

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Map

FileDownload / File: emd_6846.map.gz / Format: CCP4 / Size: 4.5 GB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationVolta phase contrast cryotomogram of a cryosectioned prometaphase S. pombe cell, strain nda3-KM311
Voxel sizeX=Y=Z: 4.6 Å
Density
Minimum - Maximum-4594 - 3240
Average (Standard dev.)169.5901 (±295.75626)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin0-901112
Dimensions40963196184
Spacing31964096184
CellA: 14701.6 Å / B: 18841.6 Å / C: 846.39996 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z4.64.64.6
M x/y/z31964096184
origin x/y/z0.0000.0000.000
length x/y/z14701.60018841.600846.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ352352352
MAP C/R/S123
start NC/NR/NS-9010112
NC/NR/NS31964096184
D min/max/mean-4594.0003240.000169.590

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Supplemental data

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Sample components

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Entire : S. pombe cell, arrested in prometaphase

EntireName: S. pombe cell, arrested in prometaphase
Components
  • Cell: S. pombe cell, arrested in prometaphase

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Supramolecule #1: S. pombe cell, arrested in prometaphase

SupramoleculeName: S. pombe cell, arrested in prometaphase / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Schizosaccharomyces pombe (fission yeast) / Strain: nda3-KM311

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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 / Details: yeast-extract supplemented
GridModel: C-flat-1/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE
High pressure freezingInstrument: OTHER
Details: Self-pressurized freezing. The value given for _emd_high_pressure_freezing.instrument is Vitrobot cup. This is not in a list of allowed values set(['LEICA EM PACT2', 'LEICA EM PACT', 'EMS- ...Details: Self-pressurized freezing. The value given for _emd_high_pressure_freezing.instrument is Vitrobot cup. 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 protectant30% dextran
SectioningUltramicrotomy - Instrument: Leica UC7/FC7 / Ultramicrotomy - Temperature: 123 K / Ultramicrotomy - Final thickness: 100 nm
Fiducial markerManufacturer: BBI / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 30400 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus min: 0.5 µm / Nominal magnification: 18000
Specialist opticsPhase plate: VOLTA PHASE PLATE
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 14.0 µm / Number grids imaged: 1 / Number real images: 61 / Average electron dose: 1.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD (ver. 4.9) / Number images used: 61

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