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
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Structure visualization
Movie
Surface view with section colored by density value
Entire Budding yeast DASH outer-kinetochore ring complex
Entire
Name: 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
Protein
Name: Budding yeast DASH outer-kinetochore ring complex / Recombinant expression: No
Mass
Theoretical: 3.4 MDa
Source
Species: Escherichia coli BL21(DE3) (bacteria)
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Experimental details
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Sample preparation
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 pH: 6.8
Vitrification
Cryogen name: ETHANE
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Electron microscopy imaging
Experimental equipment
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)
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Image acquisition
Image acquisition
Number of digital images: 67 / Sampling size: 14 microns
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Image processing
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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