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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: EMD-6912
TitleSubtomogram average of the budding yeast DASH outer-kinetochore ring.
Map dataSubtomogram average of reconstituted DASH outer-kinetochore rings around microtubules.
Sample
  • Complex: Budding yeast DASH outer-kinetochore ring complex
Biological speciesEscherichia coli BL21(DE3) (bacteria)
Methodelectron tomography / cryo EM / Resolution: 32.0 Å
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 20, 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
  • 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

emd_6912.png

Downloads & links

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Map

FileDownload / File: emd_6912.map.gz / Format: CCP4 / Size: 11.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram average of reconstituted DASH outer-kinetochore rings around microtubules.
Voxel sizeX=Y=Z: 4.61 Å
Density
Contour LevelMovie #1: 0.12
Minimum - Maximum-0.5676256 - 1.3347437
Average (Standard dev.)-0.0019778397 (±0.1068584)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions144144144
Spacing144144144
CellA=B=C: 663.84 Å
α=β=γ: 90.0 °

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/NZ000
NX/NY/NZ192192192
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
Components
  • Complex: Budding yeast DASH outer-kinetochore ring complex

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Supramolecule #1: Budding yeast DASH outer-kinetochore ring complex

SupramoleculeName: Budding yeast DASH outer-kinetochore ring complex / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 3.4 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 6.8
Details: 20 mM sodium phosphate pH 6.8, 150 mM NaCl, 1 mM EDTA
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE
DetailsDASH complexes mixed with Taxol-stabilized microtubules
SectioningOther: NO SECTIONING
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 max: 14.0 µm / Nominal defocus min: 8.0 µm / Nominal magnification: 18000
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: 67 / Average electron dose: 1.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: eTomo (ver. 4.9) / Details: CTF compensation done in Etomo
Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 32.0 Å / Resolution method: FSC 0.143 CUT-OFF
Software:
Namedetails
IMOD (ver. 4.9)Tomogram reconstruction
PEET (ver. 1.11)Template matching
RELION (ver. 2.0)Classification & refinement

Number images used: 66

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