Journal: 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
Deposition
Jan 3, 2014
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Header (metadata) release
Jan 22, 2014
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Map release
Feb 26, 2014
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Update
May 21, 2014
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Current status
May 21, 2014
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Entire : KMN network reconstituted as follows: human Mis12complex bound to...
Entire
Name: 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...
Supramolecule
Name: 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
UniProtKB: Protein MIS12 homolog / GO: MIS12/MIND type complex / InterPro: Centromere protein Mis12
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Macromolecule #2: Polyamine-modulated factor 1
Macromolecule
Name: 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
UniProtKB: Polyamine-modulated factor 1 / GO: MIS12/MIND type complex / InterPro: Kinetochore-associated protein Nnf1
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Macromolecule #3: Dsn1
Macromolecule
Name: 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
UniProtKB: Kinetochore-associated protein DSN1 homolog / GO: MIS12/MIND type complex / InterPro: Kinetochore-associated protein Dsn1/Mis13
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Macromolecule #4: Nsl1
Macromolecule
Name: 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
UniProtKB: Kinetochore-associated protein NSL1 homolog / GO: MIS12/MIND type complex / InterPro: Kinetochore Mis14/Nsl1
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Macromolecule #5: Bub-linking kinetochore protein
Macromolecule
Name: 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
Name: 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
UniProtKB: Kinetochore protein NDC80 homolog / GO: Ndc80 complex / InterPro: Kinetochore protein Ndc80
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Macromolecule #7: Spc25
Macromolecule
Name: 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
UniProtKB: Kinetochore protein Spc25 / GO: Ndc80 complex / InterPro: Chromosome segregation protein Spc25, C-terminal
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Macromolecule #8: Spc24
Macromolecule
Name: 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
Name: 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
Tilt 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.
Final reconstruction
Applied 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
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