+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-13151 | |||||||||
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Title | Negative stain EM 3D Reconstruction of the Dam1cBim1p complex. | |||||||||
Map data | A Dam1 complex dimer with an attached "arch" of one Bim1p. This sample was crosslinked with glutaraldehyde. | |||||||||
Sample |
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Function / homology | Function and homology information protein localization to microtubule plus-end / mitotic spindle polar microtubule / mitotic spindle orientation checkpoint signaling / 2-micrometer plasmid partitioning / DASH complex / protein transport along microtubule to mitotic spindle pole body / mitotic sister chromatid biorientation / positive regulation of attachment of spindle microtubules to kinetochore / mitotic spindle pole body / mitotic spindle midzone ...protein localization to microtubule plus-end / mitotic spindle polar microtubule / mitotic spindle orientation checkpoint signaling / 2-micrometer plasmid partitioning / DASH complex / protein transport along microtubule to mitotic spindle pole body / mitotic sister chromatid biorientation / positive regulation of attachment of spindle microtubules to kinetochore / mitotic spindle pole body / mitotic spindle midzone / attachment of spindle microtubules to kinetochore / nuclear migration along microtubule / protein localization to microtubule / microtubule plus-end / negative regulation of microtubule depolymerization / microtubule plus-end binding / microtubule depolymerization / microtubule organizing center / mitotic sister chromatid cohesion / spindle assembly / regulation of microtubule polymerization or depolymerization / spindle midzone / cytoplasmic microtubule / positive regulation of microtubule polymerization / spindle microtubule / mitotic spindle / spindle pole / microtubule / cell division / identical protein binding / cytoplasm Similarity search - Function | |||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 35.0 Å | |||||||||
Authors | Engelhard L / Bourque CB / Klink BU / Gatsogiannis C | |||||||||
Funding support | Germany, 1 items
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Citation | 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_13151.map.gz | 422.6 KB | EMDB map data format | |
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Header (meta data) | emd-13151-v30.xml emd-13151.xml | 18.8 KB 18.8 KB | Display Display | EMDB header |
Images | emd_13151.png | 12.1 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-13151 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13151 | HTTPS FTP |
-Related structure data
Related structure data | C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_13151.map.gz / Format: CCP4 / Size: 1.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | A Dam1 complex dimer with an attached "arch" of one Bim1p. This sample was crosslinked with glutaraldehyde. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 4.92 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
+Entire : Dam1pBim1p Complex
+Supramolecule #1: Dam1pBim1p Complex
+Macromolecule #1: Ask1 protein of the Dam1c from S. cerevisiae
+Macromolecule #2: Dam1 protein of the Dam1c from S. cerevisiae
+Macromolecule #3: Dam1 protein of the Dam1c from S. cerevisiae
+Macromolecule #4: Duo1 protein of the Dam1c from S. cerevisiae
+Macromolecule #5: Spc19 protein of the Dam1c from S. cerevisiae
+Macromolecule #6: Dad2p of the Dam1c from S. cerevisiae
+Macromolecule #7: Dad1p of the Dam1c from S. cerevisiae
+Macromolecule #8: Dad3p of the Dam1c from S. cerevisiae
+Macromolecule #9: Dad4p of the Dam1c from S. cerevisiae
+Macromolecule #10: Hsk3p of the Dam1c from S. cerevisiae
+Macromolecule #11: Bim1p of S. cerevisiae
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.01 mg/mL |
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Buffer | pH: 7.4 Component: (Formula: HEPES, NaClSodium chloride, 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 |
-Electron microscopy
Microscope | JEOL 1400 |
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Electron beam | Acceleration voltage: 120 kV / Electron source: LAB6 |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 3.4 mm |
Sample stage | Specimen holder model: JEOL |
Image recording | Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 5.0 e/Å2 |
-Image processing
Startup model | Type of model: INSILICO MODEL In silico model: Initial Model was computed using SPHIRE (VIPER module) |
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Initial angle assignment | Type: NOT APPLICABLE |
Final angle assignment | Type: NOT APPLICABLE |
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 |