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Yorodumi- EMDB-1371: Architecture of the Dam1 kinetochore ring complex and implication... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-1371 | |||||||||
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Title | Architecture of the Dam1 kinetochore ring complex and implications for microtubule-driven assembly and force-coupling mechanisms. | |||||||||
Map data | This is the reconstruction of Dam1 helical spiral around MT | |||||||||
Sample |
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Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 30.0 Å | |||||||||
Authors | Wang H-W / Ramey VH / Westermann S / Leschziner AE / Welburn JPI / Nakajima Y / Drubin DG / Barnes G / Nogales E | |||||||||
Citation | Journal: Nat Struct Mol Biol / Year: 2007 Title: Architecture of the Dam1 kinetochore ring complex and implications for microtubule-driven assembly and force-coupling mechanisms. Authors: Hong-Wei Wang / Vincent H Ramey / Stefan Westermann / Andres E Leschziner / Julie P I Welburn / Yuko Nakajima / David G Drubin / Georjana Barnes / Eva Nogales / Abstract: The Dam1 kinetochore complex is essential for chromosome segregation in budding yeast. This ten-protein complex self-assembles around microtubules, forming ring-like structures that move with ...The Dam1 kinetochore complex is essential for chromosome segregation in budding yeast. This ten-protein complex self-assembles around microtubules, forming ring-like structures that move with depolymerizing microtubule ends, a mechanism with implications for cellular function. Here we used EM-based single-particle and helical analyses to define the architecture of the Dam1 complex at 30-A resolution and the self-assembly mechanism. Ring oligomerization seems to be facilitated by a conformational change upon binding to microtubules, suggesting that the Dam1 ring is not preformed, but self-assembles around kinetochore microtubules. The C terminus of the Dam1p protein, where most of the Aurora kinase Ipl1 phosphorylation sites reside, is in a strategic location to affect oligomerization and interactions with the microtubule. One of Ipl1's roles might be to fine-tune the coupling of the microtubule interaction with the conformational change required for oligomerization, with phosphorylation resulting in ring breakdown. | |||||||||
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_1371.map.gz | 3.2 MB | EMDB map data format | |
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Header (meta data) | emd-1371-v30.xml emd-1371.xml | 11.1 KB 11.1 KB | Display Display | EMDB header |
Images | 1371.gif | 100.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-1371 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-1371 | HTTPS FTP |
-Validation report
Summary document | emd_1371_validation.pdf.gz | 291.6 KB | Display | EMDB validaton report |
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Full document | emd_1371_full_validation.pdf.gz | 290.7 KB | Display | |
Data in XML | emd_1371_validation.xml.gz | 3.1 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1371 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1371 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_1371.map.gz / Format: CCP4 / Size: 47.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | This is the reconstruction of Dam1 helical spiral around MT | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 4 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 : Dam1 complex
Entire | Name: Dam1 complex |
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Components |
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-Supramolecule #1000: Dam1 complex
Supramolecule | Name: Dam1 complex / type: sample / ID: 1000 Details: The well ordered long helical particles are rare while short helical assemblies with order are quite normal. Oligomeric state: Each assymmetric unit contains 10 subunits Number unique components: 1 |
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Molecular weight | Theoretical: 200 KDa |
-Macromolecule #1: Dam1 complex
Macromolecule | Name: Dam1 complex / type: protein_or_peptide / ID: 1 / Name.synonym: DASH / Number of copies: 1 / Oligomeric state: decamer / Recombinant expression: Yes |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast / Location in cell: kinetochore |
Molecular weight | Experimental: 200 KDa / Theoretical: 200 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | helical reconstruction |
Aggregation state | helical array |
-Sample preparation
Concentration | 1 mg/mL |
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Buffer | pH: 6.8 Details: 150 mM KCl, 20 mM potassium phosphate pH 6.8, 1 mM EDTA |
Grid | Details: 400 Quantifoil |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 100 K / Instrument: OTHER Details: Vitrification instrument: Vitrobot. 3.5 micro-liter of sample solution was applied to glow-discharged Quantifoil and blotted with filter paper for 1.5 seconds and plunged Method: Blot for 1.5 seconds before plunging |
Details | helical crystal grown in solution |
-Electron microscopy
Microscope | FEI/PHILIPS CM200FEG |
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Temperature | Average: 100 K |
Alignment procedure | Legacy - Astigmatism: objective lens astigmatism was corrected at |
Details | Low Dose mode |
Date | Nov 1, 2005 |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 12.7 µm / Number real images: 100 / Average electron dose: 15 e/Å2 / Details: Scanned on Nikon Super Coolscan 8000 / Od range: 1.5 / Bits/pixel: 14 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.6 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder: Side-entry liquid nitrogen cooled cryo-holder Specimen holder model: GATAN LIQUID NITROGEN |
-Image processing
Details | helices were formed around microtubules |
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Final reconstruction | Applied symmetry - Helical parameters - Δz: 10.2 Å Applied symmetry - Helical parameters - Δ&Phi: 24.7 ° Applied symmetry - Helical parameters - Axial symmetry: D2 (2x2 fold dihedral) Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 30.0 Å / Resolution method: OTHER / Software - Name: SUPRIM, MRC Details: Final map was calculated from two particle images. The two images were of different helical families, thus the average in little g space was performed. |