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- EMDB-2549: 3D structure of the KMN network -

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Basic information

Entry
Database: EMDB / ID: EMD-2549
Title3D structure of the KMN network
Map dataReconstruction of KMN network
Sample
  • Sample: KMN network reconstituted as follows: human Mis12complex bound to Ndc80complex (bonsai) and Knl1( from amino acids, 2106-2316)
  • Protein or peptide: Mis12
  • Protein or peptide: Polyamine-modulated factor 1
  • Protein or peptide: Dsn1
  • Protein or peptide: Nsl1
  • Protein or peptide: Bub-linking kinetochore protein
  • Protein or peptide: Highly expressed in cancer-1
  • Protein or peptide: Spc25
  • Protein or peptide: Spc24
  • Protein or peptide: Cell division cycle-assoiciated protein1
KeywordsMitosis / outer kinetochore / KMN network
Function / homology
Function and homology information


G2/MI transition of meiotic cell cycle / kinetochore adaptor activity / Knl1/Spc105 complex / positive regulation of meiosis I spindle assembly checkpoint / homologous chromosome orientation in meiotic metaphase I / MIS12/MIND type complex / skeletal muscle satellite cell proliferation / Ndc80 complex / leucine zipper domain binding / kinetochore organization ...G2/MI transition of meiotic cell cycle / kinetochore adaptor activity / Knl1/Spc105 complex / positive regulation of meiosis I spindle assembly checkpoint / homologous chromosome orientation in meiotic metaphase I / MIS12/MIND type complex / skeletal muscle satellite cell proliferation / Ndc80 complex / leucine zipper domain binding / kinetochore organization / metaphase chromosome alignment / acrosome assembly / positive regulation of mitotic cell cycle spindle assembly checkpoint / 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 / kinetochore assembly / spindle assembly involved in female meiosis I / protein localization to kinetochore / 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 / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Deposition of new CENPA-containing nucleosomes at the centromere / Resolution of Sister Chromatid Cohesion / cyclin binding / mitotic spindle organization / acrosomal vesicle / chromosome segregation / RHO GTPases Activate Formins / regulation of protein stability / kinetochore / fibrillar center / spindle pole / Separation of Sister Chromatids / azurophil granule lumen / mitotic cell cycle / microtubule binding / transcription regulator complex / transcription by RNA polymerase II / transcription coactivator activity / nuclear body / nuclear speck / cell division / intracellular membrane-bounded organelle / centrosome / Neutrophil degranulation / protein-containing complex binding / nucleolus / Golgi apparatus / extracellular region / nucleoplasm / membrane / identical protein binding / nucleus / cytosol
Similarity search - Function
Kinetochore-associated protein Nnf1 / Knl1, C-terminal RWD domain / Knl1 RWD C-terminal domain / Chromosome segregation protein Spc25, C-terminal / Kinetochore Mis14/Nsl1 / Chromosome segregation protein Spc25, C-terminal / Kinetochore Mis14/Nsl1 / Kinetochore scaffold 1 / KNL1 MELT repeat / Kinetochore protein Spc25 ...Kinetochore-associated protein Nnf1 / Knl1, C-terminal RWD domain / Knl1 RWD C-terminal domain / Chromosome segregation protein Spc25, C-terminal / Kinetochore Mis14/Nsl1 / Chromosome segregation protein Spc25, C-terminal / Kinetochore Mis14/Nsl1 / Kinetochore scaffold 1 / KNL1 MELT repeat / Kinetochore protein Spc25 / Chromosome segregation protein Spc25 / Kinetochore protein Mis14 like / MELT motif / Centromere protein Mis12 / Nuclear MIS12/MIND complex subunit PMF1/Nnf1 / Centromere protein Mis12 / Nnf1 / Mis12 protein / Kinetochore-associated protein Dsn1/Mis13 / Kinetochore-associated protein Dsn1/Mis13 / Mis12-Mtw1 protein family / Kinetochore protein Nuf2, N-terminal / Kinetochore protein Nuf2, N-terminal / Nuf2, N-terminal domain superfamily / Nuf2 family / Kinetochore protein Ndc80 / Kinetochore protein Ndc80 / Ndc80 domain superfamily / Domain of unknown function DUF5595 / HEC/Ndc80p family / Domain of unknown function (DUF5595) / Kinetochore-Ndc80 subunit Spc24 / Kinetochore-Ndc80 subunit Spc24 / Spc24 subunit of Ndc80
Similarity search - Domain/homology
Kinetochore protein NDC80 homolog / Polyamine-modulated factor 1 / Kinetochore protein Spc24 / Kinetochore scaffold 1 / Kinetochore-associated protein NSL1 homolog / Kinetochore protein Nuf2 / Protein MIS12 homolog / Kinetochore-associated protein DSN1 homolog / Kinetochore protein Spc25
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 26.7 Å
AuthorsPetrovic A / Mosalaganti S / Keller J / Mattiuzzo M / Overlack K / Krenn V / De Antoni A / Wohlgemuth S / Cecatiello V / Pasqualato S ...Petrovic A / Mosalaganti S / Keller J / Mattiuzzo M / Overlack K / Krenn V / De Antoni A / Wohlgemuth S / Cecatiello V / Pasqualato S / Raunser S / Musacchio A
CitationJournal: Mol Cell / Year: 2014
Title: Modular assembly of RWD domains on the Mis12 complex underlies outer kinetochore organization.
Authors: Arsen Petrovic / Shyamal Mosalaganti / Jenny Keller / Marta Mattiuzzo / Katharina Overlack / Veronica Krenn / Anna De Antoni / Sabine Wohlgemuth / Valentina Cecatiello / Sebastiano ...Authors: Arsen Petrovic / Shyamal Mosalaganti / Jenny Keller / Marta Mattiuzzo / Katharina Overlack / Veronica Krenn / Anna De Antoni / Sabine Wohlgemuth / Valentina Cecatiello / Sebastiano Pasqualato / Stefan Raunser / Andrea Musacchio /
Abstract: Faithful chromosome segregation is mandatory for cell and organismal viability. Kinetochores, large protein assemblies embedded in centromeric chromatin, establish a mechanical link between ...Faithful chromosome segregation is mandatory for cell and organismal viability. Kinetochores, large protein assemblies embedded in centromeric chromatin, establish a mechanical link between chromosomes and spindle microtubules. The KMN network, a conserved 10-subunit kinetochore complex, harbors the microtubule-binding interface. RWD domains in the KMN subunits Spc24 and Spc25 mediate kinetochore targeting of the microtubule-binding subunits by interacting with the Mis12 complex, a KMN subcomplex that tethers directly onto the underlying chromatin layer. Here, we show that Knl1, a KMN subunit involved in mitotic checkpoint signaling, also contains RWD domains that bind the Mis12 complex and that mediate kinetochore targeting of Knl1. By reporting the first 3D electron microscopy structure of the KMN network, we provide a comprehensive framework to interpret how interactions of RWD-containing proteins with the Mis12 complex shape KMN network topology. Our observations unveil a regular pattern in the construction of the outer kinetochore.
History
DepositionJan 3, 2014-
Header (metadata) releaseJan 22, 2014-
Map releaseFeb 26, 2014-
UpdateMay 21, 2014-
Current statusMay 21, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 9.84
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 9.84
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2549.tif

Downloads & links

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Map

FileDownload / File: emd_2549.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of KMN network
Voxel sizeX=Y=Z: 4.6 Å
Density
Contour LevelBy AUTHOR: 9.84 / Movie #1: 9.84
Minimum - Maximum-11.614444730000001 - 38.082832340000003
Average (Standard dev.)0.03312875 (±1.02653921)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 588.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.64.64.6
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z588.800588.800588.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-207-207-206
NX/NY/NZ414414414
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-11.61438.0830.033

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Supplemental data

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Segmentation: Binary mask representing the whole structure

AnnotationBinary mask representing the whole structure
Fileemd_2549_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : KMN network reconstituted as follows: human Mis12complex bound to...

EntireName: KMN network reconstituted as follows: human Mis12complex bound to Ndc80complex (bonsai) and Knl1( from amino acids, 2106-2316)
Components
  • Sample: KMN network reconstituted as follows: human Mis12complex bound to Ndc80complex (bonsai) and Knl1( from amino acids, 2106-2316)
  • Protein or peptide: Mis12
  • Protein or peptide: Polyamine-modulated factor 1
  • Protein or peptide: Dsn1
  • Protein or peptide: Nsl1
  • Protein or peptide: Bub-linking kinetochore protein
  • Protein or peptide: Highly expressed in cancer-1
  • Protein or peptide: Spc25
  • Protein or peptide: Spc24
  • Protein or peptide: Cell division cycle-assoiciated protein1

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Supramolecule #1000: KMN network reconstituted as follows: human Mis12complex bound to...

SupramoleculeName: KMN network reconstituted as follows: human Mis12complex bound to Ndc80complex (bonsai) and Knl1( from amino acids, 2106-2316)
type: sample / ID: 1000 / Details: The sample was monodisperse
Oligomeric state: One heterotetramer of Mis12C binds heterotetramer of Ndc80C and Knl1(2106-2316)
Number unique components: 9
Molecular weightExperimental: 200 KDa / Theoretical: 200 KDa / Method: Size-exclusion chromatography

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Macromolecule #1: Mis12

MacromoleculeName: Mis12 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: nucleus, centeromere / Location in cell: Kinetochore
Molecular weightExperimental: 20 KDa / Theoretical: 20 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)-codon-plus-RIL / Recombinant plasmid: pST39
SequenceUniProtKB: Protein MIS12 homolog / GO: MIS12/MIND type complex / InterPro: Centromere protein Mis12

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Macromolecule #2: Polyamine-modulated factor 1

MacromoleculeName: Polyamine-modulated factor 1 / type: protein_or_peptide / ID: 2 / Name.synonym: PMF1 or NNF1
Details: truncated protein from amino acids (31-205) was generated and used for the complex formation
Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: nucleus, centeromere / Location in cell: Kinetochore
Molecular weightExperimental: 20 KDa / Theoretical: 20 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)-codon-plus-RIL / Recombinant plasmid: pST39
SequenceUniProtKB: Polyamine-modulated factor 1 / GO: MIS12/MIND type complex / InterPro: Kinetochore-associated protein Nnf1

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Macromolecule #3: Dsn1

MacromoleculeName: Dsn1 / type: protein_or_peptide / ID: 3
Details: truncated protein from amino acids (68-356) was generated and used for the complex formation
Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: nucleus, centeromere / Location in cell: Kinetochore
Molecular weightExperimental: 40 KDa / Theoretical: 40 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)-codon-plus-RIL / Recombinant plasmid: pST39
SequenceUniProtKB: Kinetochore-associated protein DSN1 homolog / GO: MIS12/MIND type complex / InterPro: Kinetochore-associated protein Dsn1/Mis13

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Macromolecule #4: Nsl1

MacromoleculeName: Nsl1 / type: protein_or_peptide / ID: 4 / Name.synonym: Mis14, DC8, C1orf48
Details: truncated protein from amino acids (1-206) was generated and used for the complex formation
Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: nucleus, centeromere / Location in cell: Kinetochore
Molecular weightExperimental: 20 KDa / Theoretical: 20 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)-codon-plus-RIL / Recombinant plasmid: pST39
SequenceUniProtKB: Kinetochore-associated protein NSL1 homolog / GO: MIS12/MIND type complex / InterPro: Kinetochore Mis14/Nsl1

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Macromolecule #5: Bub-linking kinetochore protein

MacromoleculeName: Bub-linking kinetochore protein / type: protein_or_peptide / ID: 5 / Name.synonym: Blinkin, Casc5, Knl1
Details: truncated protein from amino acids (2106-2311) was generated and used for the complex formation
Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: nucleus, centeromere / Location in cell: Kinetochore
Molecular weightExperimental: 30 KDa / Theoretical: 30 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)-codon-plus-RIL / Recombinant plasmid: pST39
SequenceUniProtKB: Kinetochore scaffold 1

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Macromolecule #6: Highly expressed in cancer-1

MacromoleculeName: Highly expressed in cancer-1 / type: protein_or_peptide / ID: 6 / Name.synonym: Hec1, Ndc80
Details: truncated protein from amino acids (1-286) was generated and used for the complex formation
Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: nucleus, centeromere / Location in cell: Kinetochore
Molecular weightExperimental: 25 KDa / Theoretical: 25 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pGEX6P-2rbs
SequenceUniProtKB: Kinetochore protein NDC80 homolog / GO: Ndc80 complex / InterPro: Kinetochore protein Ndc80

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Macromolecule #7: Spc25

MacromoleculeName: Spc25 / type: protein_or_peptide / ID: 7 / Name.synonym: spc25
Details: truncated protein from amino acids (118-224) was generated and used for the complex formation
Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: nucleus, centeromere / Location in cell: Kinetochore
Molecular weightExperimental: 10 KDa / Theoretical: 10 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pGEX6P-2rbs
SequenceUniProtKB: Kinetochore protein Spc25 / GO: Ndc80 complex / InterPro: Chromosome segregation protein Spc25, C-terminal

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Macromolecule #8: Spc24

MacromoleculeName: Spc24 / type: protein_or_peptide / ID: 8
Details: truncated protein from amino acids (122-197) was generated and used for the complex formation
Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: nucleus, centeromere / Location in cell: Kinetochore
Molecular weightExperimental: 9 KDa / Theoretical: 9 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pGEX6P-2rbs
SequenceUniProtKB: Kinetochore protein Spc24 / GO: Ndc80 complex / InterPro: Kinetochore-Ndc80 subunit Spc24

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Macromolecule #9: Cell division cycle-assoiciated protein1

MacromoleculeName: Cell division cycle-assoiciated protein1 / type: protein_or_peptide / ID: 9 / Name.synonym: Nuf2
Details: truncated protein from amino acids (1-169) was generated and used for the complex formation
Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: nucleus, centeromere / Location in cell: Kinetochore
Molecular weightExperimental: 20 KDa / Theoretical: 20 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pGEX6P-2rbs
SequenceUniProtKB: Kinetochore protein Nuf2 / GO: Ndc80 complex / InterPro: Kinetochore protein Nuf2, N-terminal

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 8 / Details: 20mM Tris-HCl, 150mM NaCl, 1mM TCEP
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on 0.07% Uranyl formate
GridDetails: 200 mesh copper grids with thin carbon support, glow discharged
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 1400
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 67200 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: JEOL / Tilt angle min: -50
DetailsMinimal Dose system
DateOct 14, 2013
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 248 / Average electron dose: 19 e/Å2
Tilt angle max0

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Image processing

Final two d classificationNumber classes: 10
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 26.7 Å / Resolution method: OTHER / Software - Name: EMAN2/sparx / Number images used: 3764
DetailsTilt pairs were collected at 50 and 0 degrees. Particle pairs were manually selected using e2RCTboxer program. RCT reconstruction of tilted particles was calculated from best class average by back projection and was followed by back projection refinement.

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Atomic model buiding 1

Initial modelPDB ID:

2ve7


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: C
SoftwareName: Chimera
DetailsThe crystal structures were fit into the overall density manually.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

4nf9


Chain - Chain ID: A
SoftwareName: Chimera
DetailsThe crystal structures were fit into the overall density manually.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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