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- PDB-9s53: Cryo-EM structure of the base of the Saccharomyces cerevisiae KMN... -

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Basic information

Entry
Database: PDB / ID: 9s53
TitleCryo-EM structure of the base of the Saccharomyces cerevisiae KMN junction complex containing the Mis12c(Mtw1c) head 2 domain
Components
  • Kinetochore-associated protein DSN1
  • Kinetochore-associated protein MTW1
  • Kinetochore-associated protein NNF1
  • Kinetochore-associated protein NSL1
KeywordsCELL CYCLE / Kinetochore / chromosome segregation / mitosis
Function / homology
Function and homology information


MIS12/MIND type complex / spindle attachment to meiosis I kinetochore / attachment of spindle microtubules to kinetochore / meiotic sister chromatid cohesion, centromeric / kinetochore assembly / outer kinetochore / protein localization to kinetochore / mitotic sister chromatid segregation / Neutrophil degranulation / chromosome segregation ...MIS12/MIND type complex / spindle attachment to meiosis I kinetochore / attachment of spindle microtubules to kinetochore / meiotic sister chromatid cohesion, centromeric / kinetochore assembly / outer kinetochore / protein localization to kinetochore / mitotic sister chromatid segregation / Neutrophil degranulation / chromosome segregation / kinetochore / spindle pole / cell division / nucleus
Similarity search - Function
Kinetochore-associated protein Nnf1 / Kinetochore Mis14/Nsl1 / Kinetochore protein Mis14 like / Nuclear MIS12/MIND complex subunit PMF1/Nnf1 / Centromere protein Mis12 / Nnf1 / Mis12 protein / Kinetochore-associated protein Dsn1/Mis13 / Mis12-Mtw1 protein family / EF-Hand 1, calcium-binding site
Similarity search - Domain/homology
Kinetochore-associated protein MTW1 / Kinetochore-associated protein DSN1 / Kinetochore-associated protein NNF1 / Kinetochore-associated protein NSL1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.5 Å
AuthorsTurner, N.N. / Barford, D.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Cancer Research UKC576/A25675 United Kingdom
Cancer Research UKC576/A14109 United Kingdom
UK Research and Innovation (UKRI)MC_UP_1201/6 United Kingdom
CitationJournal: J Cell Biol / Year: 2026
Title: Assembly and phosphoregulatory mechanisms of the budding yeast outer kinetochore KMN complex.
Authors: Noah N Turner / Ziguo Zhang / Jing Yang / Kyle W Muir / Stephen H McLaughlin / Tomos Morgan / David Barford /
Abstract: During mitosis and meiosis, kinetochores mediate interactions between chromosomes and spindle microtubules. Kinetochores are multi-megadalton protein complexes essential for chromosome segregation; ...During mitosis and meiosis, kinetochores mediate interactions between chromosomes and spindle microtubules. Kinetochores are multi-megadalton protein complexes essential for chromosome segregation; however, recent structural, functional, and evolutionary studies have revealed divergent mechanisms of kinetochore assembly. Here, we use cryo-EM to understand the structural mechanisms by which the budding yeast microtubule-binding outer kinetochore KMN complex assembles, and how its interactions with the centromere-binding inner kinetochore are regulated. The KMN complex comprises three subcomplexes: Knl1c, Mis12cMtw1c, and Ndc80c. We show how C-terminal motifs of the Mis12cMtw1c subunits Dsn1, Mis12Mtw1, and Nnf1 bind Knl1c and Ndc80c. At the opposite end of the Mis12cMtw1c stalk, an N-terminal auto-inhibitory segment of Dsn1 (Dsn1AI) folds into two α-helices that engage the Mis12cMtw1c head 1 domain, thereby occluding binding sites for the inner kinetochore subunits CENP-CMif2 and CENP-UAme1, reducing their affinity for Mis12cMtw1. Our structure reveals how Aurora BIpl1 phosphorylation of Dsn1AI would release this auto-inhibition to substantially strengthen preexisting connections between the inner and outer kinetochore.
History
DepositionJul 29, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 8, 2026Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
Ds: Kinetochore-associated protein DSN1
Mt: Kinetochore-associated protein MTW1
Nn: Kinetochore-associated protein NNF1
Ns: Kinetochore-associated protein NSL1


Theoretical massNumber of molelcules
Total (without water)148,1854
Polymers148,1854
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Kinetochore-associated protein DSN1


Mass: 65777.023 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: DSN1, YIR010W, YIB10W / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P40568
#2: Protein Kinetochore-associated protein MTW1 / Mis12-like protein


Mass: 33291.180 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / Gene: MTW1, DSN3, NSL2, YAL034W-A, YAL034AW / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P39731
#3: Protein Kinetochore-associated protein NNF1


Mass: 23668.541 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / Gene: NNF1, YJR112W, J2011 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P47149
#4: Protein Kinetochore-associated protein NSL1


Mass: 25448.398 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: S288c / Gene: NSL1, YPL233W / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q12143
Has protein modificationN

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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

Component
IDNameTypeDetails (eV)Entity IDParent-IDSource
1Saccharomyces cerevisiae outer kinetochore KMN junction complexCOMPLEXImaged sample also contained Saccharomyces cerevisiae kinetochore protein NDC80 (UniProtKB accession P40460); Saccharomyces cerevisiae kinetochore protein NUF2 (UniProtKB accession P33895) with a C-terminal HRV-3C protease cleavage site and twin-strep-tag II affinity tag; Saccharomyces cerevisiae kinetochore protein SPC24 (UniProtKB accession code Q04477); Saccharomyces cerevisiae Kinetochore protein SPC25 (UniProtKB accession code P40014); Saccharomyces cerevisiae outer kinetochore KNL1 complex subunit SPC105 (UniProtKB accession code P53148); and Saccharomyces cerevisiae outer kinetochore KNL1 complex subunit KRE28 (UniProtKB accession code Q04431) with a C-terminal TEV protease cleavage site and twin-strep-tag II affinity tagall0RECOMBINANT
2Saccharomyces cerevisiae outer kinetochore Mis12c(Mtw1c) subcomplexCOMPLEXall1RECOMBINANT
3Saccharomyces cerevisiae outer kinetochore Knl1c subcomplexCOMPLEXN-terminal truncation of Knl1(Spc105) residues 1-4441RECOMBINANT
4Saccharomyces cerevisiae outer kinetochore Ndc80cCOMPLEXall1RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
11438.876 kDa/nmNO
21147.937 kDa/nmYES
31103.894 kDa/nmNO
41187.080 kDa/nmYES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
21Saccharomyces cerevisiae (brewer's yeast)4932288c
32Saccharomyces cerevisiae (brewer's yeast)4932288c
43Saccharomyces cerevisiae (brewer's yeast)4932288c
54Saccharomyces cerevisiae (brewer's yeast)4932288c
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
21Trichoplusia ni (cabbage looper)7111High five
32Trichoplusia ni (cabbage looper)7111High five
43Trichoplusia ni (cabbage looper)7111High five
54Trichoplusia ni (cabbage looper)7111High five
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
2150 mMSodium chlorideNaCl1
31 mMTCEP1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1400 nm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.75 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 26390
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
7UCSF ChimeraXmodel fitting
8Cootmodel fitting
10RELION5initial Euler assignment
12RELION5classification
13RELION53D reconstruction
14PHENIX1.21.2_5419:model refinement
CTF correctionDetails: CTFFIND 4.1 used as implemented in Relion 4.0 / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2592458
Details: Selected using Topaz particle picker trained on manually picked micrographs
3D reconstructionResolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 18160
Details: Multibody refinement in RELION 5.0 using Blush regularization
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDChain-IDChain residue rangeSource nameTypeAccession codePdb chain residue range
1Dsn1229-365AlphaFoldin silico model
2Nsl12-96AlphaFoldin silico model
39S4Q

9s4q

Mtw1Mtw11-151PDBexperimental model9S4Q1-151
49S4Q

9s4q

Nnf1Nnf11-153PDBexperimental model9S4Q1-153
59S4Q

9s4q

Dsn1Dsn1435-483PDBexperimental model9S4Q435-483
69S4Q

9s4q

Nsl1Nsl197-159PDBexperimental model9S4Q97-159

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