|Entry||Database: EMDB / ID: 6914|
|Title||Serial cryotomogram of a metaphase budding yeast cell.|
|Map data||Serial cryotomogram of a metaphase budding yeast cell|
|Sample||S. cerevisiae cell, arrested in metaphase:|
|Source||Saccharomyces cerevisiae (baker's yeast)|
|Method||electron tomography / cryo EM|
|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 22, 2018 / Header (metadata) release: Nov 28, 2018 / Map release: Nov 28, 2018 / Last update: Nov 28, 2018|
Downloads & links
|File||emd_6914.map.gz (map file in CCP4 format, 12279765 KB)|
|Voxel size||X=Y=Z: 8.93 Å|
CCP4 map header:
-Entire S. cerevisiae cell, arrested in metaphase
|Entire||Name: S. cerevisiae cell, arrested in metaphase / Number of components: 1|
-Component #1: cellular-component, S. cerevisiae cell, arrested in metaphase
|Cellular-component||Name: S. cerevisiae cell, arrested in metaphase / Recombinant expression: No|
|Source||Species: Saccharomyces cerevisiae (baker's yeast) / Strain: W303|
|Specimen||Specimen state: cell / Method: cryo EM|
|Sample solution||Buffer solution: YEPD / pH: 7|
|Support film||plasma cleaned at 15 mA for 45 seconds, carbon side pre-coated with BSA-Gold mixture|
|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: 1.6 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 8700.0 X (nominal), 15678.0 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 10000.0 - nm|
|Specimen Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Camera||Detector: FEI FALCON II (4k x 4k)|
|Image acquisition||Number of digital images: 61 / Sampling size: 14 microns|
|Processing||Method: electron tomography / Number of sections: 61|
|3D reconstruction||Algorithm: BACK PROJECTION / Software: IMOD / CTF correction: Phase flipping done in Etomo 4.10|
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