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

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Basic information

Entry
Database: EMDB / ID: 6914
TitleSerial cryotomogram of a metaphase budding yeast cell.
Map dataSerial cryotomogram of a metaphase budding yeast cell
SampleS. cerevisiae cell, arrested in metaphase:
SourceSaccharomyces cerevisiae (baker'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: 2018
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.
DateDeposition: Feb 22, 2018 / Header (metadata) release: Nov 28, 2018 / Map release: Nov 28, 2018 / Last update: Nov 28, 2018

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Structure visualization

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  • Imaged by UCSF Chimera
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Supplemental images

Downloads & links

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Map

Fileemd_6914.map.gz (map file in CCP4 format, 12279765 KB)
Voxel sizeX=Y=Z: 8.93 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)-16.042185 (10.322338999999999)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions43343268867
Origin-184.0-1239.0102.0
Limit4149.02028.0968.0
Spacing32684334867
CellA: 29183.24 Å / B: 38702.62 Å / C: 7742.31 Å
α=β=γ: 90.0 deg.

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/NZ
NX/NY/NZ
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 / Number of components: 1

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Component #1: cellular-component, S. cerevisiae cell, arrested in metaphase

Cellular-componentName: S. cerevisiae cell, arrested in metaphase / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast) / Strain: W303

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Experimental details

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Sample preparation

SpecimenSpecimen state: cell / Method: cryo EM
Sample solutionBuffer solution: YEPD / pH: 7
Support filmplasma cleaned at 15 mA for 45 seconds, carbon side pre-coated with BSA-Gold mixture
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.6 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 8700.0 X (nominal), 15678.0 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 10000.0 - nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: FEI FALCON II (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 61 / Sampling size: 14 microns

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Image processing

ProcessingMethod: electron tomography / Number of sections: 61
3D reconstructionAlgorithm: BACK PROJECTION / Software: IMOD / CTF correction: Phase flipping done in Etomo 4.10

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