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- EMDB-5254: WT Dam1 complex assembled into a ring around a microtubule -

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Basic information

Entry
Database: EMDB / ID: EMD-5254
TitleWT Dam1 complex assembled into a ring around a microtubule
Map data16 WT Dam1 complexes assembled into a ring around a microtubule
Sample
  • Sample: WT Dam1 ring
  • Protein or peptide: Dam1 complex
KeywordsKinetochore / MAP
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 35.0 Å
AuthorsRamey V / Wang HW / Nogales E
CitationJournal: Mol Biol Cell / Year: 2011
Title: The Dam1 ring binds to the E-hook of tubulin and diffuses along the microtubule.
Authors: Vincent H Ramey / Hong-Wei Wang / Yuko Nakajima / Amanda Wong / Jian Liu / David Drubin / Georjana Barnes / Eva Nogales /
Abstract: There has been much effort in recent years aimed at understanding the molecular mechanism by which the Dam1 kinetochore complex is able to couple microtubule depolymerization to poleward movement. ...There has been much effort in recent years aimed at understanding the molecular mechanism by which the Dam1 kinetochore complex is able to couple microtubule depolymerization to poleward movement. Both a biased diffusion and a forced walk model have been proposed, and several key functional aspects of Dam1-microtubule binding are disputed. Here, we investigate the elements involved in tubulin-Dam1 complex interactions and directly visualize Dam1 rings on microtubules in order to infer their dynamic behavior on the microtubule lattice and its likely relevance at the kinetochore. We find that the Dam1 complex has a preference for native tubulin over tubulin that is lacking its acidic C-terminal tail. Statistical mechanical analysis of images of Dam1 rings on microtubules, applied to both the distance between rings and the tilt angle of the rings with respect to the microtubule axis, supports a diffusive ring model. We also present a cryo-EM reconstruction of the Dam1 ring, likely the relevant assembly form of the complex for energy coupling during microtubule depolymerization in budding yeast. The present studies constitute a significant step forward by linking structural and biochemical observations toward a comprehensive understanding of the Dam1 complex.
History
DepositionDec 10, 2010-
Header (metadata) releaseFeb 1, 2011-
Map releaseFeb 1, 2011-
UpdateJul 17, 2013-
Current statusJul 17, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.29
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.29
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5254_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5254.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation16 WT Dam1 complexes assembled into a ring around a microtubule
Voxel sizeX=Y=Z: 4.6 Å
Density
Contour LevelBy AUTHOR: 1.29 / Movie #1: 1.29
Minimum - Maximum-3.1440239 - 4.78089142
Average (Standard dev.)0.00002663 (±0.24046032)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 920.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.64.64.6
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z920.000920.000920.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-3.1444.7810.000

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Supplemental data

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Sample components

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Entire : WT Dam1 ring

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

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Supramolecule #1000: WT Dam1 ring

SupramoleculeName: WT Dam1 ring / type: sample / ID: 1000 / Oligomeric state: 16 Dam1 complexes / Number unique components: 1
Molecular weightTheoretical: 210 KDa

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Macromolecule #1: Dam1 complex

MacromoleculeName: Dam1 complex / type: protein_or_peptide / ID: 1 / Name.synonym: Dam1 complex / Number of copies: 16 / Oligomeric state: Ring / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Budding Yeast / Location in cell: Nucleus
Molecular weightTheoretical: 210 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 6.8 / Details: 80mM PIPES, 1mM EGTA, 1mM MgCl2
GridDetails: 400 mesh C-flat 1.2um holes
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 77 K / Instrument: OTHER
Details: Vitrification instrument: FEI vitrobot. Waited for sample to pass through open holes before blotting
Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.6 mm / Nominal defocus max: 2.8 µm / Nominal defocus min: 1.17 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Single tilt cryo / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 91 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 100,000 times magnification
DateMar 31, 2009
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 50 / Average electron dose: 15 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: per micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 35.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER
Details: 16 fold rotational symmetry applied to reconstruction
Number images used: 236

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