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- EMDB-6914: Serial cryotomogram of a metaphase budding yeast cell. -

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Basic information

Entry
Database: EMDB / ID: EMD-6914
TitleSerial cryotomogram of a metaphase budding yeast cell.
Map dataSerial cryotomogram of a metaphase budding yeast cell
Sample
  • Cell: S. cerevisiae cell, arrested in metaphase
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodelectron tomography / cryo EM
AuthorsNg C / Deng L / Chen C / Lim H / Shi J / Surana U / Gan L
CitationJournal: J Cell Biol / Year: 2019
Title: Electron cryotomography analysis of Dam1C/DASH at the kinetochore-spindle interface in situ.
Authors: Cai Tong Ng / Li Deng / Chen Chen / Hong Hwa Lim / Jian Shi / Uttam Surana / Lu Gan /
Abstract: In dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at their kinetochores. While kinetochore subcomplexes have been studied extensively in vitro, little is known about ...In dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at their kinetochores. While kinetochore subcomplexes have been studied extensively in vitro, little is known about their in vivo structure and interactions with microtubules or their response to spindle damage. Here we combine electron cryotomography of serial cryosections with genetic and pharmacological perturbation to study the yeast chromosome segregation machinery in vivo. Each kinetochore microtubule has one (rarely, two) Dam1C/DASH outer kinetochore assemblies. Dam1C/DASH contacts the microtubule walls and does so with its flexible "bridges"; there are no contacts with the protofilaments' curved tips. In metaphase, ∼40% of the Dam1C/DASH assemblies are complete rings; the rest are partial rings. Ring completeness and binding position along the microtubule are sensitive to kinetochore attachment and tension, respectively. Our study and those of others support a model in which each kinetochore must undergo cycles of conformational change to couple microtubule depolymerization to chromosome movement.
History
DepositionFeb 22, 2018-
Header (metadata) releaseNov 28, 2018-
Map releaseNov 28, 2018-
UpdateJun 12, 2019-
Current statusJun 12, 2019Processing 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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Movie viewer
Supplemental images

emd_6914.png

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Map

FileDownload / File: emd_6914.map.gz / Format: CCP4 / Size: 11.4 GB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationSerial cryotomogram of a metaphase budding yeast cell
Voxel sizeX=Y=Z: 8.93 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)-16.042185 (±10.322338999999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-184-1239102
Dimensions43343268867
Spacing32684334867
CellA: 29183.24 Å / B: 38702.62 Å / C: 7742.31 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z8.938.93000023073378.93
M x/y/z32684334867
origin x/y/z0.0000.0000.000
length x/y/z29183.24038702.6217742.310
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ192192192
MAP C/R/S123
start NC/NR/NS-1239-184102
NC/NR/NS32684334867
D min/max/mean-128.000127.000-16.042

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Supplemental data

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Sample components

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Entire : S. cerevisiae cell, arrested in metaphase

EntireName: S. cerevisiae cell, arrested in metaphase
Components
  • Cell: S. cerevisiae cell, arrested in metaphase

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Supramolecule #1: S. cerevisiae cell, arrested in metaphase

SupramoleculeName: S. cerevisiae cell, arrested in metaphase / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: W303

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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: YEPD
GridModel: C-flat-2/0.5 4C / Material: COPPER / Mesh: 150 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: AIR
Details: plasma cleaned at 15 mA for 45 seconds, carbon side pre-coated with BSA-Gold mixture
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 protectant25% 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: 15678 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus min: 10.0 µm / Nominal magnification: 8700
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 exposure time: 1.0 sec. / Average electron dose: 1.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: eTomo (ver. 4.10) / Details: Phase flipping done in Etomo 4.10
Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD (ver. 4.10) / Number images used: 61

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