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- PDB-3iz0: Human Ndc80 Bonsai Decorated Microtubule -

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Basic information

Entry
Database: PDB / ID: 3iz0
TitleHuman Ndc80 Bonsai Decorated Microtubule
Components
  • NDC80-SPC25 chimera protein, Chain B from PDB 2VE7 (Ndc80 bonsai)
  • NUF2-SPC24 chimera protein, Chain D from PDB 2VE7 (Ndc80 bonsai)
  • alpha tubulin, Chain A from PDB 1JFF
  • beta tubulin, Chain B from PDB 1JFF
KeywordsCELL CYCLE / Ndc80 / 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 / cytoplasmic microtubule / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / 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
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / TAXOL / 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 speciesHomo sapiens (human)
Bos taurus (cattle)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 8.6 Å
AuthorsAlushin, G.M. / Ramey, V.H. / Pasqualato, S. / Ball, D.A. / Grigorieff, N. / Musacchio, A. / Nogales, E.
CitationJournal: 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.
History
DepositionAug 9, 2010Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 13, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Jun 19, 2013Group: Database references
Revision 1.3Jul 18, 2018Group: Author supporting evidence / Data collection
Category: em_image_scans / em_single_particle_entity / em_software
Item: _em_software.image_processing_id / _em_software.name
Revision 1.4Feb 21, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: alpha tubulin, Chain A from PDB 1JFF
B: beta tubulin, Chain B from PDB 1JFF
C: NDC80-SPC25 chimera protein, Chain B from PDB 2VE7 (Ndc80 bonsai)
D: NUF2-SPC24 chimera protein, Chain D from PDB 2VE7 (Ndc80 bonsai)
E: NDC80-SPC25 chimera protein, Chain B from PDB 2VE7 (Ndc80 bonsai)
F: NUF2-SPC24 chimera protein, Chain D from PDB 2VE7 (Ndc80 bonsai)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)232,61411
Polymers230,7046
Non-polymers1,9105
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
DetailsAUTHORS STATE THAT THE NDC80 COMPLEX FORMS OLIGOMERS ALONG MICROTUBULE PROTOFILAMENTS, BINDING ONCE PER TUBULIN MONOMER. THIS ENTRY CONSISTS OF COORDINATES FOR ONE TUBULIN HETERODIMER BOUND TO A CLUSTER OF 2 NDC80 MOLECULES. THE BINDING SITE IS FORMED BETWEEN TUBULIN MONOMERS, AND THUS THERE IS ONE COMPLETE NDC80-TUBULIN INTERFACE AND ONE NDC80-NDC80 INTERFACE. LARGER CLUSTERS ARE MORE PREVALENT IN VITRO AND ARE LIKELY TO EXIST IN VIVO.

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Components

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Protein , 4 types, 6 molecules ABCEDF

#1: Protein alpha tubulin, Chain A from PDB 1JFF


Mass: 50107.238 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Cellular location: cytoskeleton / Tissue: brain / References: UniProt: P81947*PLUS
#2: Protein beta tubulin, Chain B from PDB 1JFF


Mass: 49907.770 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Cellular location: cytoskeleton / Tissue: brain / References: UniProt: Q6B856*PLUS
#3: Protein NDC80-SPC25 chimera protein, Chain B from PDB 2VE7 (Ndc80 bonsai)


Mass: 36351.297 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NDC80 SPC25, SPBC25, AD024 / Plasmid: pGEX6p-2RBS / Production host: Escherichia coli (E. coli)
References: UniProt: Q05DQ6, UniProt: Q9HBM1, UniProt: O14777*PLUS
#4: Protein NUF2-SPC24 chimera protein, Chain D from PDB 2VE7 (Ndc80 bonsai)


Mass: 28993.418 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NUF2, RP11-77M5.2-004, SPC24 / Plasmid: pGEX6p-2RBS / Production host: Escherichia coli (E. coli)
References: UniProt: B1AQT4, UniProt: C9JGC4, UniProt: Q9BZD4*PLUS

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Non-polymers , 5 types, 5 molecules

#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#7: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#8: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine diphosphate


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#9: Chemical ChemComp-TA1 / TAXOL / Paclitaxel


Mass: 853.906 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C47H51NO14 / Comment: medication, chemotherapy*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeDetailsParent-ID
1Human Ndc80 bonsai complex bound to the microtubuleCOMPLEX2 copies of the Ndc80 bonsai complex bind to each tubulin heterodimer. Ndc80 bonsai is a heterodimer of Ndc80-Spc25 and Nuf2-Spc24 Tubulin is a heterodimer of alpha-beta tubulin0
2tubulin1
3Ndc80-Spc25 Chimera1
4Nuf2-Spc24 Chimera1
Molecular weightValue: 0.26 MDa / Experimental value: NO
Buffer solutionpH: 6.8
Details: 80mM PIPES, 1mM MgCl2, 1mM EGTA, 1mM DTT, 0.05% Nonidet P-40, 20uM taxol
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 80mM PIPES, 1mM MgCl2, 1mM EGTA, 1mM DTT, 0.05% Nonidet P-40, 20uM taxol
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 %
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 imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Mar 12, 2009
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm / Cs: 2.2 mm
Astigmatism: objective lens astigmatism corrected at 100Kx mag
Camera length: 0 mm
Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN / Specimen holder type: side-entry / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 15 e/Å2 / Film or detector model: KODAK SO-163 FILM
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1UCSF Chimeramodel fitting
2FREALIGN3D reconstruction
3SPIDER3D reconstruction
3D reconstructionMethod: IRSHR / Resolution: 8.6 Å / Resolution method: FSC 0.143 CUT-OFF
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.
Symmetry type: HELICAL
Atomic model building
IDProtocolSpaceDetails
1RIGID BODY FITREALREFINEMENT PROTOCOL--Rigid Body
2RIGID BODY FITREALREFINEMENT PROTOCOL--Rigid Body
3RIGID BODY FITREALREFINEMENT PROTOCOL--Rigid Body
4RIGID BODY FITREALREFINEMENT PROTOCOL--Rigid Body
Atomic model building

Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-ID 3D fitting-IDAccession codeInitial refinement model-ID
11JFF

1jff

A11JFF1
21JFF

1jff

B21JFF1
32VE7

2ve7

B32VE72
42VE7

2ve7

D42VE72
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms12030 0 124 0 12154

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