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- EMDB-6912: Subtomogram average of the budding yeast DASH outer-kinetochore ring. -

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Basic information

Entry
Database: EMDB / ID: 6912
TitleSubtomogram average of the budding yeast DASH outer-kinetochore ring.
Map dataSubtomogram average of reconstituted DASH outer-kinetochore rings around microtubules.
SampleBudding yeast DASH outer-kinetochore ring complex:
SourceEscherichia coli BL21(DE3) (bacteria)
Methodelectron tomography / cryo EM / 32 Å resolution
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 20, 2018 / Header (metadata) release: Nov 28, 2018 / Map release: Nov 28, 2018 / Last update: Nov 28, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_6912.map.gz (map file in CCP4 format, 11944 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
144 pix
4.61 Å/pix.
= 663.84 Å
144 pix
4.61 Å/pix.
= 663.84 Å
144 pix
4.61 Å/pix.
= 663.84 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.61 Å
Density
Contour Level:0.12 (movie #1):
Minimum - Maximum-0.5676256 - 1.3347437
Average (Standard dev.)-0.0019778397 (0.1068584)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions144144144
Origin0.00.00.0
Limit143.0143.0143.0
Spacing144144144
CellA=B=C: 663.84 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.614.614.61
M x/y/z144144144
origin x/y/z0.0000.0000.000
length x/y/z663.840663.840663.840
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS144144144
D min/max/mean-0.5681.335-0.002

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Supplemental data

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Sample components

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Entire Budding yeast DASH outer-kinetochore ring complex

EntireName: Budding yeast DASH outer-kinetochore ring complex / Number of components: 1

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Component #1: protein, Budding yeast DASH outer-kinetochore ring complex

ProteinName: Budding yeast DASH outer-kinetochore ring complex / Recombinant expression: No
MassTheoretical: 3.4 MDa
SourceSpecies: Escherichia coli BL21(DE3) (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 1 mg/ml
Buffer solution: 20 mM sodium phosphate pH 6.8, 150 mM NaCl, 1 mM EDTA
pH: 6.8
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: 1.5 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 18000.0 X (nominal), 30400.0 X (calibrated) / Imaging mode: BRIGHT FIELD / Defocus: 8000.0 - 14000.0 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: 67 / Sampling size: 14 microns

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Image processing

ProcessingMethod: electron tomography / Number of sections: 66
3D reconstructionAlgorithm: BACK PROJECTION / Software: IMOD, PEET, RELION / CTF correction: CTF compensation done in Etomo / Resolution: 32 Å / Resolution method: FSC 0.143 CUT-OFF

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