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- EMDB-1371: Architecture of the Dam1 kinetochore ring complex and implication... -

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Basic information

Entry
Database: EMDB / ID: EMD-1371
TitleArchitecture of the Dam1 kinetochore ring complex and implications for microtubule-driven assembly and force-coupling mechanisms.
Map dataThis is the reconstruction of Dam1 helical spiral around MT
Sample
  • Sample: Dam1 complex
  • Protein or peptide: Dam1 complex
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodhelical reconstruction / cryo EM / Resolution: 30.0 Å
AuthorsWang H-W / Ramey VH / Westermann S / Leschziner AE / Welburn JPI / Nakajima Y / Drubin DG / Barnes G / Nogales E
CitationJournal: 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
DepositionJun 12, 2007-
Header (metadata) releaseJun 13, 2007-
Map releaseJun 13, 2007-
UpdateApr 13, 2016-
Current statusApr 13, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 10.093457944
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 10.093457944
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1371.gif

Downloads & links

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Map

FileDownload / File: emd_1371.map.gz / Format: CCP4 / Size: 47.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is the reconstruction of Dam1 helical spiral around MT
Voxel sizeX=Y=Z: 4 Å
Density
Contour Level1: 6.99 / Movie #1: 10.0934579
Minimum - Maximum-35.0 - 38.0
Average (Standard dev.)-0.129246 (±5.13426)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions251251201
Spacing251251201
CellA: 1004 Å / B: 1004 Å / C: 804 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z444
M x/y/z251251201
origin x/y/z0.0000.0000.000
length x/y/z1004.0001004.000804.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS251251201
D min/max/mean-35.00038.000-0.129

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Supplemental data

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Sample components

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Entire : Dam1 complex

EntireName: Dam1 complex
Components
  • Sample: Dam1 complex
  • Protein or peptide: Dam1 complex

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Supramolecule #1000: Dam1 complex

SupramoleculeName: 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
Molecular weightTheoretical: 200 KDa

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Macromolecule #1: Dam1 complex

MacromoleculeName: Dam1 complex / type: protein_or_peptide / ID: 1 / Name.synonym: DASH / Number of copies: 1 / Oligomeric state: decamer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast / Location in cell: kinetochore
Molecular weightExperimental: 200 KDa / Theoretical: 200 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration1 mg/mL
BufferpH: 6.8
Details: 150 mM KCl, 20 mM potassium phosphate pH 6.8, 1 mM EDTA
GridDetails: 400 Quantifoil
VitrificationCryogen 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
Detailshelical crystal grown in solution

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.6 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Side-entry liquid nitrogen cooled cryo-holder
Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 100 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at
DetailsLow Dose mode
DateNov 1, 2005
Image recordingCategory: 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

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Image processing

Final reconstructionApplied 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.
Detailshelices were formed around microtubules

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