|Entry||Database: EMDB / ID: 6912|
|Title||Subtomogram average of the budding yeast DASH outer-kinetochore ring.|
|Map data||Subtomogram average of reconstituted DASH outer-kinetochore rings around microtubules.|
|Sample||Budding yeast DASH outer-kinetochore ring complex:|
|Source||Escherichia coli BL21(DE3) (bacteria)|
|Method||electron tomography / cryo EM / 32 Å resolution|
|Authors||Ng C / Deng L / Chen C / Lim H / Shi J / Surana U / Gan L|
|Citation||Journal: 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.
|Date||Deposition: Feb 20, 2018 / Header (metadata) release: Nov 28, 2018 / Map release: Nov 28, 2018 / Last update: Nov 28, 2018|
|Structure viewer||EM map: |
Downloads & links
|File||emd_6912.map.gz (map file in CCP4 format, 11944 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 4.61 Å|
CCP4 map header:
-Entire Budding yeast DASH outer-kinetochore ring complex
|Entire||Name: Budding yeast DASH outer-kinetochore ring complex / Number of components: 1|
-Component #1: protein, Budding yeast DASH outer-kinetochore ring complex
|Protein||Name: Budding yeast DASH outer-kinetochore ring complex / Recombinant expression: No|
|Mass||Theoretical: 3.4 MDa|
|Source||Species: Escherichia coli BL21(DE3) (bacteria)|
|Specimen||Specimen state: particle / Method: cryo EM|
|Sample solution||Specimen conc.: 1 mg/ml|
Buffer solution: 20 mM sodium phosphate pH 6.8, 150 mM NaCl, 1 mM EDTA
|Vitrification||Cryogen name: ETHANE|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.5 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 18000.0 X (nominal), 30400.0 X (calibrated) / Imaging mode: BRIGHT FIELD / Defocus: 8000.0 - 14000.0 nm|
|Specimen Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Camera||Detector: FEI FALCON II (4k x 4k)|
|Image acquisition||Number of digital images: 67 / Sampling size: 14 microns|
|Processing||Method: electron tomography / Number of sections: 66|
|3D reconstruction||Algorithm: 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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