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- PDB-8ppr: Structure of the human outer kinetochore KMN network complex -

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Basic information

Entry
Database: PDB / ID: 8ppr
TitleStructure of the human outer kinetochore KMN network complex
Components
  • (Kinetochore protein ...) x 2
  • (Kinetochore-associated protein ...) x 2
  • Kinetochore scaffold 1
  • Polyamine-modulated factor 1
  • Protein MIS12 homolog
  • ZW10 interactor
KeywordsCELL CYCLE / Kinetochore / complex / KMN
Function / homology
Function and homology information


regulation of meiosis I spindle assembly checkpoint / 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 / acrosome assembly / regulation of mitotic cell cycle spindle assembly checkpoint ...regulation of meiosis I spindle assembly checkpoint / 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 / acrosome assembly / regulation of mitotic cell cycle spindle assembly checkpoint / attachment of spindle microtubules to kinetochore / attachment of mitotic spindle microtubules to kinetochore / kinetochore assembly / protein localization to kinetochore / mitotic spindle assembly checkpoint signaling / mitotic sister chromatid segregation / 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 / mitotic spindle organization / acrosomal vesicle / chromosome segregation / RHO GTPases Activate Formins / establishment of localization in cell / kinetochore / fibrillar center / spindle pole / Separation of Sister Chromatids / azurophil granule lumen / transcription regulator complex / transcription by RNA polymerase II / transcription coactivator activity / nuclear body / nuclear speck / cell division / intracellular membrane-bounded organelle / dendrite / Neutrophil degranulation / nucleolus / Golgi apparatus / extracellular region / nucleoplasm / nucleus / cytosol / cytoplasm
Similarity search - Function
ZW10 interactor / ZW10 interactor / Knl1, C-terminal RWD domain / Knl1 RWD C-terminal domain / Chromosome segregation protein Spc25, C-terminal / Kinetochore Mis14/Nsl1 / Kinetochore scaffold 1 / KNL1 MELT repeat / Kinetochore protein Spc25 / Chromosome segregation protein Spc25 ...ZW10 interactor / ZW10 interactor / Knl1, C-terminal RWD domain / Knl1 RWD C-terminal domain / 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 / Nuclear MIS12/MIND complex subunit PMF1/Nnf1 / Centromere protein Mis12 / Nnf1 / Mis12 protein / Kinetochore-associated protein Dsn1/Mis13 / Mis12-Mtw1 protein family / Kinetochore-Ndc80 subunit Spc24 / Spc24 subunit of Ndc80
Similarity search - Domain/homology
ZW10 interactor / Polyamine-modulated factor 1 / Kinetochore protein Spc24 / Kinetochore scaffold 1 / Kinetochore-associated protein NSL1 homolog / Protein MIS12 homolog / Kinetochore-associated protein DSN1 homolog / Kinetochore protein Spc25
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsYatskevich, S. / Barford, D.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
UK Research and Innovation (UKRI)MC_UP_1201/6 United Kingdom
Cancer Research UKC576/A14109 United Kingdom
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Structure of the human outer kinetochore KMN network complex.
Authors: Stanislau Yatskevich / Jing Yang / Dom Bellini / Ziguo Zhang / David Barford /
Abstract: Faithful chromosome segregation requires robust, load-bearing attachments of chromosomes to the mitotic spindle, a function accomplished by large macromolecular complexes termed kinetochores. In most ...Faithful chromosome segregation requires robust, load-bearing attachments of chromosomes to the mitotic spindle, a function accomplished by large macromolecular complexes termed kinetochores. In most eukaryotes, the constitutive centromere-associated network (CCAN) complex of the inner kinetochore recruits to centromeres the ten-subunit outer kinetochore KMN network that comprises the KNL1C, MIS12C and NDC80C complexes. The KMN network directly attaches CCAN to microtubules through MIS12C and NDC80C. Here, we determined a high-resolution cryo-EM structure of the human KMN network. This showed an intricate and extensive assembly of KMN subunits, with the central MIS12C forming rigid interfaces with NDC80C and KNL1C, augmented by multiple peptidic inter-subunit connections. We also observed that unphosphorylated MIS12C exists in an auto-inhibited state that suppresses its capacity to interact with CCAN. Ser100 and Ser109 of the N-terminal segment of the MIS12C subunit Dsn1, two key targets of Aurora B kinase, directly stabilize this auto-inhibition. Our study indicates how selectively relieving this auto-inhibition through Ser100 and Ser109 phosphorylation might restrict outer kinetochore assembly to functional centromeres during cell division.
History
DepositionJul 8, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 20, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
D: Kinetochore-associated protein DSN1 homolog
M: Protein MIS12 homolog
N: Kinetochore-associated protein NSL1 homolog
P: Polyamine-modulated factor 1
F: Kinetochore protein Spc24
G: Kinetochore protein Spc25
K: Kinetochore scaffold 1
Z: ZW10 interactor


Theoretical massNumber of molelcules
Total (without water)465,5888
Polymers465,5888
Non-polymers00
Water181
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Kinetochore-associated protein ... , 2 types, 2 molecules DN

#1: Protein Kinetochore-associated protein DSN1 homolog


Mass: 40122.195 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DSN1, C20orf172, MIS13 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9H410
#3: Protein Kinetochore-associated protein NSL1 homolog


Mass: 32208.951 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NSL1, C1orf48, DC31, DC8, MIS14 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q96IY1

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Protein , 4 types, 4 molecules MPKZ

#2: Protein Protein MIS12 homolog


Mass: 24170.922 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MIS12 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9H081
#4: Protein Polyamine-modulated factor 1 / PMF-1


Mass: 23368.311 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PMF1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q6P1K2
#7: Protein Kinetochore scaffold 1 / ALL1-fused gene from chromosome 15q14 protein / AF15q14 / Bub-linking kinetochore protein / Blinkin ...ALL1-fused gene from chromosome 15q14 protein / AF15q14 / Bub-linking kinetochore protein / Blinkin / Cancer susceptibility candidate gene 5 protein / Cancer/testis antigen 29 / CT29 / Kinetochore-null protein 1 / Protein CASC5 / Protein D40/AF15q14


Mass: 265722.125 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KNL1, CASC5, KIAA1570 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q8NG31
#8: Protein ZW10 interactor / ZW10-interacting protein 1 / Zwint-1


Mass: 31333.359 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ZWINT / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O95229

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Kinetochore protein ... , 2 types, 2 molecules FG

#5: Protein Kinetochore protein Spc24 / / hSpc24


Mass: 22469.113 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SPC24, SPBC24 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q8NBT2
#6: Protein Kinetochore protein Spc25 / / hSpc25


Mass: 26192.832 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SPC25, SPBC25, AD024 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9HBM1

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Non-polymers , 1 types, 1 molecules

#9: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Outer kinetochore KMN network junction complex / Type: COMPLEX / Entity ID: #1-#8 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 45 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.18.2_3874: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 599831 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00312863
ELECTRON MICROSCOPYf_angle_d0.53117314
ELECTRON MICROSCOPYf_dihedral_angle_d8.6371658
ELECTRON MICROSCOPYf_chiral_restr0.0371916
ELECTRON MICROSCOPYf_plane_restr0.0042250

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