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- EMDB-1373: Architecture of the Dam1 kinetochore ring complex and implication... -

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Basic information

Entry
Database: EMDB / ID: EMD-1373
TitleArchitecture of the Dam1 kinetochore ring complex and implications for microtubule-driven assembly and force-coupling mechanisms.
Map dataThis is the single particle reconstruction of the DeltaC Dam1 mutant complex in its dimeric form.
Sample
  • Sample: Dam1 DeltC mutant complex
  • Protein or peptide: Dam1 DeltC mutant complex
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 29.0 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 13, 2007-
Header (metadata) releaseJun 13, 2007-
Map releaseJun 13, 2007-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 7.278150038
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 7.278150038
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1373.gif

Downloads & links

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Map

FileDownload / File: emd_1373.map.gz / Format: CCP4 / Size: 8.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is the single particle reconstruction of the DeltaC Dam1 mutant complex in its dimeric form.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4 Å/pix.
x 130 pix.
= 520. Å		4 Å/pix.
x 130 pix.
= 520. Å		4 Å/pix.
x 130 pix.
= 520. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 2.44 / Movie #1: 7.27815
Minimum - Maximum-5.74718 - 30.2408
Average (Standard dev.)-0.000191472 (±0.976579)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions130130130
Spacing130130130
CellA=B=C: 520 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z444
M x/y/z130130130
origin x/y/z0.0000.0000.000
length x/y/z520.000520.000520.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS130130130
D min/max/mean-5.74730.241-0.000

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Supplemental data

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Sample components

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Entire : Dam1 DeltC mutant complex

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

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Supramolecule #1000: Dam1 DeltC mutant complex

SupramoleculeName: Dam1 DeltC mutant complex / type: sample / ID: 1000
Details: The sample was mainly composed of dimers and little percentage of monomers and trimers.
Oligomeric state: Dimeric decamer / Number unique components: 1
Molecular weightExperimental: 200 KDa / Theoretical: 200 KDa

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Macromolecule #1: Dam1 DeltC mutant complex

MacromoleculeName: Dam1 DeltC mutant complex / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Oligomeric state: dimer of 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

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2 mg/mL
BufferpH: 6.8
Details: 500 mM NaCl, 20 mM sodium phosphate pH 6.8, 1 mM EDTA
StainingType: NEGATIVE
Details: The complexes were negatively stained with 2% uranyl formate with the sandwich method between two layers of thin carbon film.
GridDetails: 400 mesh copper grid
VitrificationCryogen name: NONE

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Electron microscopy

MicroscopeFEI TECNAI 12
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 120,000 magnification
DetailsThe micrographs were taken at low dose mode.
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 12.7 µm / Number real images: 200
Details: The micrographs were scanned on a Nikon Super Coolscan 8000 scanner
Od range: 1.4 / Bits/pixel: 14
Tilt angle min0
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 6.6 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 49000
Sample stageSpecimen holder: Eucentric / Specimen holder model: OTHER

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Image processing

DetailsThe particles were selected using WEB program manually.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 29.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC, SPIDER / Number images used: 5984
Final angle assignmentDetails: SPIDER: theta 90 degrees, phi 90 degrees
Final two d classificationNumber classes: 50

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