Journal: EMBO J / Year: 2021 Title: Phospho-regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore. Authors: Alexander Dudziak / Lena Engelhard / Cole Bourque / Björn Udo Klink / Pascaline Rombaut / Nikolay Kornakov / Karolin Jänen / Franz Herzog / Christos Gatsogiannis / Stefan Westermann / Abstract: Kinetochores form the link between chromosomes and microtubules of the mitotic spindle. The heterodecameric Dam1 complex (Dam1c) is a major component of the Saccharomyces cerevisiae outer ...Kinetochores form the link between chromosomes and microtubules of the mitotic spindle. The heterodecameric Dam1 complex (Dam1c) is a major component of the Saccharomyces cerevisiae outer kinetochore, assembling into 3 MDa-sized microtubule-embracing rings, but how ring assembly is specifically initiated in vivo remains to be understood. Here, we describe a molecular pathway that provides local control of ring assembly during the establishment of sister kinetochore bi-orientation. We show that Dam1c and the general microtubule plus end-associated protein (+TIP) Bim1/EB1 form a stable complex depending on a conserved motif in the Duo1 subunit of Dam1c. EM analyses reveal that Bim1 crosslinks protrusion domains of adjacent Dam1c heterodecamers and promotes the formation of oligomers with defined curvature. Disruption of the Dam1c-Bim1 interaction impairs kinetochore localization of Dam1c in metaphase and delays mitosis. Phosphorylation promotes Dam1c-Bim1 binding by relieving an intramolecular inhibition of the Dam1 C-terminus. In addition, Bim1 recruits Bik1/CLIP-170 to Dam1c and induces formation of full rings even in the absence of microtubules. Our data help to explain how new kinetochore end-on attachments are formed during the process of attachment error correction.
History
Deposition
Jun 30, 2021
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Header (metadata) release
Jul 28, 2021
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Map release
Jul 28, 2021
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Update
Sep 29, 2021
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Current status
Sep 29, 2021
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_13151.map.gz / Format: CCP4 / Size: 1.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
A Dam1 complex dimer with an attached "arch" of one Bim1p. This sample was crosslinked with glutaraldehyde.
Voxel size
X=Y=Z: 4.92 Å
Density
Contour Level
By AUTHOR: 20.0 / Movie #1: 20
Minimum - Maximum
-0.775786 - 162.91025
Average (Standard dev.)
0.9978052 (±6.8564415)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
76
76
76
Spacing
76
76
76
Cell
A=B=C: 373.92 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
4.92
4.92
4.92
M x/y/z
76
76
76
origin x/y/z
0.000
0.000
0.000
length x/y/z
373.920
373.920
373.920
α/β/γ
90.000
90.000
90.000
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
76
76
76
D min/max/mean
-0.776
162.910
0.998
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Supplemental data
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Sample components
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Entire : Dam1pBim1p Complex
Entire
Name: Dam1pBim1p Complex
Components
Complex: Dam1pBim1p Complex
Protein or peptide: Ask1 protein of the Dam1c from S. cerevisiae
Protein or peptide: Dam1 protein of the Dam1c from S. cerevisiae
Protein or peptide: Dam1 protein of the Dam1c from S. cerevisiae
Protein or peptide: Duo1 protein of the Dam1c from S. cerevisiae
Protein or peptide: Spc19 protein of the Dam1c from S. cerevisiae
Protein or peptide: Dad2p of the Dam1c from S. cerevisiae
Protein or peptide: Dad1p of the Dam1c from S. cerevisiae
Protein or peptide: Dad3p of the Dam1c from S. cerevisiae
Protein or peptide: Dad4p of the Dam1c from S. cerevisiae
Protein or peptide: Hsk3p of the Dam1c from S. cerevisiae
Protein or peptide: Bim1p of S. cerevisiae
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Supramolecule #1: Dam1pBim1p Complex
Supramolecule
Name: Dam1pBim1p Complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all Details: An unknown number of copies of Bim1p (2-4) binds to a dimeric complex formed by two Dam1c heterodecamers
pH: 7.4 / Component: (Formula: HEPES, NaCl, TCEP, Glycerol) Details: The sample was crosslinked using 0.5 % glutaraldehyde. The crosslinking reaction was stopped after 60 seconds by the addition of TRIS
Staining
Type: NEGATIVE / Material: Uranyl Formate Details: 4 microl of complex were applied on freshly glow-discharged carbon-coated copper grids (Agar Scientific, G400C). After an incubation of 2 minutes, the sample was blotted with Whatman no. 4 ...Details: 4 microl of complex were applied on freshly glow-discharged carbon-coated copper grids (Agar Scientific, G400C). After an incubation of 2 minutes, the sample was blotted with Whatman no. 4 filter paper, washed 2 times with ddH2O and stained with 0.75 % uranyl formate.
Grid
Model: Homemade / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE
Details
25 mM HEPES, pH 7.4, 200 mM NaCl, 1 mM MgCl2, 0.5 mM TCEP and 2.5 (v/v) % glycerol
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Electron microscopy
Microscope
JEOL 1400
Image recording
Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 5.0 e/Å2
Electron beam
Acceleration voltage: 120 kV / Electron source: LAB6
Electron optics
Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 3.4 mm
Sample stage
Specimen holder model: JEOL
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Image processing
Startup model
Type of model: INSILICO MODEL In silico model: Initial Model was computed using SPHIRE (VIPER module)
Final reconstruction
Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 35.0 Å / Resolution method: OTHER / Software - Name: SPHIRE / Number images used: 36980
Initial angle assignment
Type: NOT APPLICABLE
Final angle assignment
Type: NOT APPLICABLE
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