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- EMDB-5490: Reconstruction of the Wild-type Ndc80 Bonsai Decorated Microtubul... -

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Basic information

Entry
Database: EMDB / ID: EMD-5490
TitleReconstruction of the Wild-type Ndc80 Bonsai Decorated Microtubule on CCD for Difference Map Calculation
Map dataReconstruction of a 14 protofilament microtubule with wild-type ndc80 Bonsai
Sample
  • Sample: Human Ndc80 bonsai complex bound to the microtubule
  • Protein or peptide: tubulin
  • Protein or peptide: Ndc80-Spc25 Chimera
  • Protein or peptide: Nuf2-Spc24 Chimera
Keywordsmicrotubule / Ndc80 / Hec1 / kinetochore / mitosis
Biological speciesBos taurus (cattle) / Homo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 12.8 Å
AuthorsAlushin GM / Musinipally V / Matson D / Tooley J / Stukenberg PT / Nogales E
CitationJournal: Nat Struct Mol Biol / Year: 2012
Title: Multimodal microtubule binding by the Ndc80 kinetochore complex.
Authors: Gregory M Alushin / Vivek Musinipally / Daniel Matson / John Tooley / P Todd Stukenberg / Eva Nogales /
Abstract: The Ndc80 complex is a key site of kinetochore-microtubule attachment during cell division. The human complex engages microtubules with a globular 'head' formed by tandem calponin-homology domains ...The Ndc80 complex is a key site of kinetochore-microtubule attachment during cell division. The human complex engages microtubules with a globular 'head' formed by tandem calponin-homology domains and an 80-amino-acid unstructured 'tail' that contains sites of phosphoregulation by the Aurora B kinase. Using biochemical, cell biological and electron microscopy analyses, we dissected the roles of the tail in binding of microtubules and mediation of cooperative interactions between Ndc80 complexes. Two segments of the tail that contain Aurora B phosphorylation sites become ordered at interfaces; one with tubulin and the second with an adjacent Ndc80 head on the microtubule surface, forming interactions that are disrupted by phosphorylation. We propose a model in which Ndc80's interaction with either growing or shrinking microtubule ends can be tuned by the phosphorylation state of its tail.
History
DepositionSep 1, 2012-
Header (metadata) releaseSep 26, 2012-
Map releaseOct 3, 2012-
UpdateMar 26, 2014-
Current statusMar 26, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.67
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.67
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_5490.map.gz / Format: CCP4 / Size: 1.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of a 14 protofilament microtubule with wild-type ndc80 Bonsai
Voxel sizeX=Y=Z: 2.74 Å
Density
Contour LevelBy AUTHOR: 1.67 / Movie #1: 1.67
Minimum - Maximum-5.98720503 - 5.54348898
Average (Standard dev.)0.17337535 (±1.82936239)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions707070
Spacing707070
CellA=B=C: 191.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.742.742.74
M x/y/z707070
origin x/y/z0.0000.0000.000
length x/y/z191.800191.800191.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-5029166
NX/NY/NZ106122134
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS707070
D min/max/mean-5.9875.5430.173

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Supplemental data

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Sample components

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Entire : Human Ndc80 bonsai complex bound to the microtubule

EntireName: Human Ndc80 bonsai complex bound to the microtubule
Components
  • Sample: Human Ndc80 bonsai complex bound to the microtubule
  • Protein or peptide: tubulin
  • Protein or peptide: Ndc80-Spc25 Chimera
  • Protein or peptide: Nuf2-Spc24 Chimera

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Supramolecule #1000: Human Ndc80 bonsai complex bound to the microtubule

SupramoleculeName: Human Ndc80 bonsai complex bound to the microtubule / type: sample / ID: 1000
Details: Ndc80 bonsai is a heterodimer of Ndc80-Spc25 and Nuf2-Spc24; tubulin is a heterodimer of alpha and beta tubulin
Oligomeric state: 2 copies of the Ndc80 bonsai complex bind to each tubulin heterodimer
Number unique components: 2
Molecular weightTheoretical: 260 KDa

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Macromolecule #1: tubulin

MacromoleculeName: tubulin / type: protein_or_peptide / ID: 1 / Name.synonym: tubulin / Number of copies: 1 / Oligomeric state: heterodimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: bovine / Tissue: brain / Location in cell: cytoskeleton
Molecular weightTheoretical: 110 KDa

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Macromolecule #2: Ndc80-Spc25 Chimera

MacromoleculeName: Ndc80-Spc25 Chimera / type: protein_or_peptide / ID: 2
Details: Chimera of Ndc80 residues 1-286 and Spc25 residues 118-224
Number of copies: 1 / Oligomeric state: heterodimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human / Organelle: Nucleus / Location in cell: Kinetochore
Molecular weightTheoretical: 45 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pGEX6p-2RBS

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Macromolecule #3: Nuf2-Spc24 Chimera

MacromoleculeName: Nuf2-Spc24 Chimera / type: protein_or_peptide / ID: 3
Details: Chimera of Nuf2 residues 1-169 and Spc24 residues 122-197
Number of copies: 1 / Oligomeric state: heterodimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: Nucleus / Location in cell: Kinetochore
Molecular weightTheoretical: 29 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pGEX6p-2RBS

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.25 mg/mL
BufferpH: 6.8
Details: 80mM PIPES, 1mM MgCl2, 1mM EGTA, 1mM DTT, 0.05% Nonidet P-40, 20uM taxol
GridDetails: C-flat 1.2/1.3
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK II
Method: 2 uL of 0.25 mg/mL MTs applied to grid for 1 minute, 4 uL of 0.7 mg/mL Ndc80 bonsai added, manually blot 1 minute, another 4 uL of Ndc80 applied for 1 minute, 2 uL removed with pipettor, blot ...Method: 2 uL of 0.25 mg/mL MTs applied to grid for 1 minute, 4 uL of 0.7 mg/mL Ndc80 bonsai added, manually blot 1 minute, another 4 uL of Ndc80 applied for 1 minute, 2 uL removed with pipettor, blot for 2 seconds before plunging, 0 mm offset

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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: 2.2 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 80000
Sample stageSpecimen holder: side-entry / Specimen holder model: GATAN LIQUID NITROGEN
Alignment procedureLegacy - Astigmatism: objective lens astigmatism corrected at 100Kx mag
DateOct 29, 2011
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 202 / Average electron dose: 20 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Phase-flipping each image
Final reconstructionApplied symmetry - Helical parameters - Δz: 9.04016 Å
Applied symmetry - Helical parameters - Δ&Phi: 25.76246 °
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 12.8 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2/SPARX
Details: Particles were aligned using multi-model IHRSR protocol in EMAN2/SPARX with naked 13 and 14 protofilament microtubules as references. The deposited map is a segmented region for difference ...Details: Particles were aligned using multi-model IHRSR protocol in EMAN2/SPARX with naked 13 and 14 protofilament microtubules as references. The deposited map is a segmented region for difference map calculation. No amplitude scaling was applied.
DetailsThe phase-flipped particles were aligned using IHRSR in EMAN2/SPARX.

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