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- EMDB-2153: Electron tomography of Saccharomyces cerevisiae kinetochore fragm... -

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Basic information

Entry
Database: EMDB / ID: EMD-2153
TitleElectron tomography of Saccharomyces cerevisiae kinetochore fragment on a taxol stabilized microtubule
Map dataTomogram of S cerevisiae kinetochore fragment on a taxol-stabilized microtubule
Sample
  • Sample: Saccharomyces cerevisiae kinetochore fragment on a taxol stabilized microtubule
  • Organelle or cellular component: Microtubule
  • Organelle or cellular component: Kinetochore fragment
KeywordsKinetochore / microtubule / dam1 / ndc80 / KMN / taxol / Saccharomyces cerevisiae
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodelectron tomography / negative staining
AuthorsGonen S / Akiyoshi B / Iadanza MG / Shi D / Duggan N / Biggins S / Gonen T
CitationJournal: Nat Struct Mol Biol / Year: 2012
Title: The structure of purified kinetochores reveals multiple microtubule-attachment sites.
Authors: Shane Gonen / Bungo Akiyoshi / Matthew G Iadanza / Dan Shi / Nicole Duggan / Sue Biggins / Tamir Gonen /
Abstract: Chromosomes must be accurately partitioned to daughter cells to prevent aneuploidy, a hallmark of many tumors and birth defects. Kinetochores are the macromolecular machines that segregate ...Chromosomes must be accurately partitioned to daughter cells to prevent aneuploidy, a hallmark of many tumors and birth defects. Kinetochores are the macromolecular machines that segregate chromosomes by maintaining load-bearing attachments to the dynamic tips of microtubules. Here, we present the structure of isolated budding-yeast kinetochore particles, as visualized by EM and electron tomography of negatively stained preparations. The kinetochore appears as an ~126-nm particle containing a large central hub surrounded by multiple outer globular domains. In the presence of microtubules, some particles also have a ring that encircles the microtubule. Our data, showing that kinetochores bind to microtubules via multivalent attachments, lay the foundation to uncover the key mechanical and regulatory mechanisms by which kinetochores control chromosome segregation and cell division.
History
DepositionJun 29, 2012-
Header (metadata) releaseAug 15, 2012-
Map releaseAug 15, 2012-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

emd_2153.jpg

Downloads & links

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Map

FileDownload / File: emd_2153.map.gz / Format: CCP4 / Size: 97.8 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationTomogram of S cerevisiae kinetochore fragment on a taxol-stabilized microtubule
Voxel sizeX=Y=Z: 4.3 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)-84.007637020000004 (±9.687632560000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-511-51150
Dimensions10251025100
Spacing10251025100
CellA: 4407.5 Å / B: 4407.5 Å / C: 430.00003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z4.34.34.3
M x/y/z10251025100
origin x/y/z0.0000.0000.000
length x/y/z4407.5004407.500430.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-32-32-32
NX/NY/NZ646464
MAP C/R/S123
start NC/NR/NS-511-51150
NC/NR/NS10251025100
D min/max/mean-128.000127.000-84.008

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Supplemental data

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Sample components

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Entire : Saccharomyces cerevisiae kinetochore fragment on a taxol stabiliz...

EntireName: Saccharomyces cerevisiae kinetochore fragment on a taxol stabilized microtubule
Components
  • Sample: Saccharomyces cerevisiae kinetochore fragment on a taxol stabilized microtubule
  • Organelle or cellular component: Microtubule
  • Organelle or cellular component: Kinetochore fragment

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Supramolecule #1000: Saccharomyces cerevisiae kinetochore fragment on a taxol stabiliz...

SupramoleculeName: Saccharomyces cerevisiae kinetochore fragment on a taxol stabilized microtubule
type: sample / ID: 1000
Details: fragment of native kinetochore, individual components not identified
Number unique components: 2

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Supramolecule #1: Microtubule

SupramoleculeName: Microtubule / type: organelle_or_cellular_component / ID: 1 / Details: stabilized with taxol / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: SBY8253 / synonym: Baker's Yeast / Location in cell: Cytoplasm

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Supramolecule #2: Kinetochore fragment

SupramoleculeName: Kinetochore fragment / type: organelle_or_cellular_component / ID: 2 / Details: individual components not identified / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: SBY8253 / synonym: Baker's Yeast / Location in cell: Cytoplasm

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Experimental details

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Structure determination

Methodnegative staining
Processingelectron tomography

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Sample preparation

BufferpH: 8
Details: 25 mM HEPES, 0.2 mM MgCl2, 0.1 mM EDTA, 0.5 mM EGTA, 150 mM KCl, 15% (v/v) Glycerol, 0.1% (v/v) NP-40
StainingType: NEGATIVE
Details: Sample applied to grid for 20 sec, washed with one drop of ultrapure water and two drops of 0.75% uranyl formate. Anti-mouse IgG-gold fiducial markers added and second layer of carbon applied by evaporation.
GridDetails: Gilder 400 mesh copper grid, carbon coated by evaporation. Positive discharge.
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 2.0 µm
Sample stageSpecimen holder model: OTHER / Tilt series - Axis1 - Min angle: -70 ° / Tilt series - Axis1 - Max angle: 70 ° / Tilt series - Axis1 - Angle increment: 2 °
DateJan 6, 2012
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Number real images: 100

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Image processing

Final reconstructionSoftware - Name: Amira, IMOD / Number images used: 100

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