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- EMDB-5223: Human Ndc80 Bonsai Decorated Microtubule -

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Basic information

Entry
Database: EMDB / ID: EMD-5223
TitleHuman Ndc80 Bonsai Decorated Microtubule
Map dataReal space helical reconstruction of the human Ndc80 bonsai decorated microtubule
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
KeywordsNdc80 / HEC1 / NUF2 / tubulin / kinetochore / mitosis / calponin homology domain / microtubule
Function / homology
Function and homology information


G2/MI transition of meiotic cell cycle / kinetochore adaptor activity / skeletal muscle satellite cell proliferation / Ndc80 complex / kinetochore => GO:0000776 / kinetochore organization / metaphase chromosome alignment / positive regulation of mitotic cell cycle spindle assembly checkpoint / meiotic chromosome segregation / attachment of spindle microtubules to kinetochore ...G2/MI transition of meiotic cell cycle / kinetochore adaptor activity / skeletal muscle satellite cell proliferation / Ndc80 complex / kinetochore => GO:0000776 / kinetochore organization / metaphase chromosome alignment / positive regulation of mitotic cell cycle spindle assembly checkpoint / meiotic chromosome segregation / attachment of spindle microtubules to kinetochore / outer kinetochore / attachment of mitotic spindle microtubules to kinetochore / spindle assembly involved in female meiosis I / positive regulation of axon guidance / mitotic spindle assembly checkpoint signaling / mitotic sister chromatid segregation / establishment of mitotic spindle orientation / centrosome duplication / chromosome, centromeric region / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / cytoplasmic microtubule / microtubule-based process / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Resolution of Sister Chromatid Cohesion / cellular response to interleukin-4 / cyclin binding / mitotic spindle organization / chromosome segregation / RHO GTPases Activate Formins / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / regulation of protein stability / structural constituent of cytoskeleton / microtubule cytoskeleton organization / kinetochore / Separation of Sister Chromatids / microtubule cytoskeleton / double-stranded RNA binding / mitotic cell cycle / nervous system development / microtubule binding / microtubule / cell cycle / protein heterodimerization activity / cell division / GTPase activity / centrosome / ubiquitin protein ligase binding / protein-containing complex binding / GTP binding / nucleolus / nucleoplasm / membrane / identical protein binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Chromosome segregation protein Spc25, C-terminal / Kinetochore protein Spc25 / Chromosome segregation protein Spc25 / Kinetochore protein Nuf2, N-terminal / Nuf2, N-terminal domain superfamily / Nuf2 family / Kinetochore protein Ndc80 / Ndc80 domain superfamily / Domain of unknown function DUF5595 / HEC/Ndc80p family ...Chromosome segregation protein Spc25, C-terminal / Kinetochore protein Spc25 / Chromosome segregation protein Spc25 / Kinetochore protein Nuf2, N-terminal / Nuf2, N-terminal domain superfamily / Nuf2 family / Kinetochore protein Ndc80 / Ndc80 domain superfamily / Domain of unknown function DUF5595 / HEC/Ndc80p family / Domain of unknown function (DUF5595) / Kinetochore-Ndc80 subunit Spc24 / Spc24 subunit of Ndc80 / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily
Similarity search - Domain/homology
Kinetochore protein Nuf2 / Kinetochore protein Spc24 / Kinetochore protein NDC80 homolog / Tubulin alpha-1B chain / Kinetochore protein Hec1 / Tubulin beta-2B chain / Kinetochore protein Nuf2 / Kinetochore protein Spc25
Similarity search - Component
Biological speciesBos taurus (cattle) / Homo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 8.6 Å
AuthorsAlushin GM / Ramey VH / Pasqualato S / Ball DA / Grigorieff N / Musacchio A / Nogales E
Citation
Journal: Nature / Year: 2010
Title: The Ndc80 kinetochore complex forms oligomeric arrays along microtubules.
Authors: Gregory M Alushin / Vincent H Ramey / Sebastiano Pasqualato / David A Ball / Nikolaus Grigorieff / Andrea Musacchio / Eva Nogales /
Abstract: The Ndc80 complex is a key site of regulated kinetochore-microtubule attachment (a process required for cell division), but the molecular mechanism underlying its function remains unknown. Here we ...The Ndc80 complex is a key site of regulated kinetochore-microtubule attachment (a process required for cell division), but the molecular mechanism underlying its function remains unknown. Here we present a subnanometre-resolution cryo-electron microscopy reconstruction of the human Ndc80 complex bound to microtubules, sufficient for precise docking of crystal structures of the component proteins. We find that the Ndc80 complex binds the microtubule with a tubulin monomer repeat, recognizing α- and β-tubulin at both intra- and inter-tubulin dimer interfaces in a manner that is sensitive to tubulin conformation. Furthermore, Ndc80 complexes self-associate along protofilaments through interactions mediated by the amino-terminal tail of the NDC80 protein, which is the site of phospho-regulation by Aurora B kinase. The complex's mode of interaction with the microtubule and its oligomerization suggest a mechanism by which Aurora B could regulate the stability of load-bearing kinetochore-microtubule attachments.
#1: Journal: CELL (CAMBRIDGE,MASS.) / Year: 2008
Title: Implications for kinetochore-microtubule attachment from the structure of an engineered Ndc80 complex
Authors: Ciferri C / Pasqualato S / Screpanti E / Varetti G / Santaguida S / Dos Reis G / Maiolica A / Polka J / De Luca JG / De Wulf P / Salek M / Rappsilber J / Moores CA / Salmon ED / Musacchio A
#2: Journal: J.MOL.BIOL. / Year: 2001
Title: Refined structure of alpha-beta tubulin from zinc-induced sheets stabilized with taxol
Authors: Lowe J / Li H / Downing KH / Nogales E
History
DepositionAug 4, 2010-
Header (metadata) releaseAug 13, 2010-
Map releaseOct 4, 2010-
UpdateOct 19, 2010-
Current statusOct 19, 2010Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.9
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.9
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3iz0
  • Surface level: 1.9
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3iz0
  • Surface level: 1.9
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3iz0
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5223_1.png

Downloads & links

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Map

FileDownload / File: emd_5223.map.gz / Format: CCP4 / Size: 18 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReal space helical reconstruction of the human Ndc80 bonsai decorated microtubule
Voxel sizeX=Y=Z: 2.48 Å
Density
Contour LevelBy AUTHOR: 1.9 / Movie #1: 1.9
Minimum - Maximum-4.59434 - 6.14608
Average (Standard dev.)0.00000000640655 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220100
Spacing220220100
CellA: 545.6 Å / B: 545.6 Å / C: 248 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.482.482.48
M x/y/z220220100
origin x/y/z0.0000.0000.000
length x/y/z545.600545.600248.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-99-99-99
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220100
D min/max/mean-4.5946.1460.000

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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-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: Cow / 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 / Name.synonym: Ndc80-Spc25 Chimera
Details: Chimera of Ndc80 residues 1-286 with 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 / Name.synonym: Nuf2-Spc24 Chimera
Details: Chimera of Nuf2 residues 1-169 with 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: OTHER / Details: Vitrification instrument: Vitrobot
Method: 2ul of 0.25 mg per ml MTs applied to grid for 1 minute 4ul of 0.7 mg per ml Ndc80 bonsai added, 1 minute manually blotted, then another 4ul of Ndc80 applied 1 minute 2ul removed with pipetter ...Method: 2ul of 0.25 mg per ml MTs applied to grid for 1 minute 4ul of 0.7 mg per ml Ndc80 bonsai added, 1 minute manually blotted, then another 4ul of Ndc80 applied 1 minute 2ul removed with pipetter Blot for 2 seconds before plunging, 0mm offset

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 50000
Sample stageSpecimen holder: side-entry / Specimen holder model: GATAN LIQUID NITROGEN
Alignment procedureLegacy - Astigmatism: objective lens astigmatism corrected at 100Kx mag
DateMar 12, 2009
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.35 µm / Number real images: 100 / Average electron dose: 15 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 9.54113 Å
Applied symmetry - Helical parameters - Δ&Phi: 27.67984 °
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Spider, FREALIGN
Details: Particles were initially aligned using IHRSR protocol in SPIDER with naked MT as reference. Final reconstruction and CTF correction was performed with FREALIGN. A B-factor of -450 was ...Details: Particles were initially aligned using IHRSR protocol in SPIDER with naked MT as reference. Final reconstruction and CTF correction was performed with FREALIGN. A B-factor of -450 was applied with BFACTOR. FSC was calculated only for MT and Ndc80-NUF2 head, disordered outer head was excluded with soft mask.

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Atomic model buiding 1

Initial modelPDB ID:

1jff

SoftwareName: Chimera
DetailsProtocol: Rigid Body
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3iz0:
Human Ndc80 Bonsai Decorated Microtubule

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Atomic model buiding 2

Initial modelPDB ID:

2ve7


Chain - Chain ID: B
SoftwareName: Chimera
DetailsPDBEntryID_givenInChain. Protocol: Rigid Body. The pdb was manually edited to remove disordered residues in EM map it ends at residue four hundred thirty seven
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3iz0:
Human Ndc80 Bonsai Decorated Microtubule

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Atomic model buiding 3

Initial modelPDB ID:

2ve7


Chain - Chain ID: D
SoftwareName: Chimera
DetailsPDBEntryID_givenInChain. Protocol: Rigid Body. The pdb was manually edited to remove disordered residues in EM map it ends at residue one hundred fifty six
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3iz0:
Human Ndc80 Bonsai Decorated Microtubule

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