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- PDB-9s5n: Cryo-EM structure of the Saccharomyces cerevisiae KMN junction co... -

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Basic information

Entry
Database: PDB / ID: 9s5n
TitleCryo-EM structure of the Saccharomyces cerevisiae KMN junction complex containing the Mis12c(Mtw1c) head 2 domain
Components
  • (Kinetochore protein ...) x 2
  • (Kinetochore-associated protein ...) x 4
  • (Outer kinetochore KNL1 complex subunit ...) x 2
KeywordsCELL CYCLE / Kinetochore / chromosome segregation / mitosis
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 / centromere clustering / MIS12/MIND type complex / Ndc80 complex / mitotic sister chromatid biorientation / spindle attachment to meiosis I kinetochore / sister chromatid biorientation ...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 / centromere clustering / MIS12/MIND type complex / Ndc80 complex / mitotic sister chromatid biorientation / spindle attachment to meiosis I kinetochore / sister chromatid biorientation / attachment of spindle microtubules to kinetochore / meiotic sister chromatid cohesion, centromeric / kinetochore assembly / outer kinetochore / protein localization to kinetochore / mitotic spindle assembly checkpoint signaling / mitotic sister chromatid segregation / chromosome, centromeric region / Neutrophil degranulation / chromosome segregation / kinetochore / spindle pole / microtubule binding / cell division / mitochondrion / identical protein binding / nucleus
Similarity search - Function
Spindle pole body component Kre28 / Spindle pole body component Kre28 / Spc7 kinetochore protein domain / Spc105/Spc7 / Spc7 kinetochore protein / Spc7 kinetochore protein / Kinetochore-associated protein Nnf1 / Spc24, Fungi, globular domain superfamily / Knl1, C-terminal RWD domain / Knl1 RWD C-terminal domain ...Spindle pole body component Kre28 / Spindle pole body component Kre28 / Spc7 kinetochore protein domain / Spc105/Spc7 / Spc7 kinetochore protein / Spc7 kinetochore protein / Kinetochore-associated protein Nnf1 / Spc24, Fungi, globular domain superfamily / Knl1, C-terminal RWD domain / Knl1 RWD C-terminal domain / 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 / Kinetochore-Ndc80 subunit Spc24 / Spc24 subunit of Ndc80 / EF-Hand 1, calcium-binding site
Similarity search - Domain/homology
Kinetochore-associated protein MTW1 / Kinetochore protein SPC25 / Kinetochore-associated protein DSN1 / Kinetochore-associated protein NNF1 / Outer kinetochore KNL1 complex subunit SPC105 / Outer kinetochore KNL1 complex subunit KRE28 / Kinetochore protein SPC24 / Kinetochore-associated protein NSL1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Saccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.2 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
Revision 1.0Apr 8, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 8, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Apr 8, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 8, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 8, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 8, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Apr 22, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.1Apr 22, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
Nn: Kinetochore-associated protein NNF1
Mt: Kinetochore-associated protein MTW1
Zw: Outer kinetochore KNL1 complex subunit KRE28
Kl: Outer kinetochore KNL1 complex subunit SPC105
Ds: Kinetochore-associated protein DSN1
24: Kinetochore protein SPC24
25: Kinetochore protein SPC25
Ns: Kinetochore-associated protein NSL1


Theoretical massNumber of molelcules
Total (without water)302,1558
Polymers302,1558
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

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Kinetochore-associated protein ... , 4 types, 4 molecules NnMtDsNs

#1: 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 (brewer's yeast)
Gene: NNF1, YJR112W, J2011 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P47149
#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) / Gene: MTW1, DSN3, NSL2, YAL034W-A, YAL034AW / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P39731
#5: 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
#8: 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) / Gene: NSL1, YPL233W / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q12143

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Outer kinetochore KNL1 complex subunit ... , 2 types, 2 molecules ZwKl

#3: Protein Outer kinetochore KNL1 complex subunit KRE28 / Spindle pole body component KRE28


Mass: 48468.273 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: KRE28, YDR532C / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q04431
#4: Protein Outer kinetochore KNL1 complex subunit SPC105 / 105 kDa spindle pole component protein / Spindle pole body component SPC105


Mass: 55581.047 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: SPC105, KNL1, YGL093W / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P53148

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Kinetochore protein ... , 2 types, 2 molecules 2425

#6: Protein Kinetochore protein SPC24


Mass: 24639.814 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: SPC24, YMR117C, YM9718.16C / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q04477
#7: Protein Kinetochore protein SPC25


Mass: 25280.326 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: SPC25, YER018C / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P40014

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Details

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), and Saccharomyces cerevisiae kinetochore protein NUF2 (UniProtKB accession P33895) with a C-terminal HRV-3C protease cleavage site and twin-strep-tag II affinity tag.all0RECOMBINANT
2Saccharomyces cerevisiae outer kinetochore Mis12c(Mtw1c) subcomplexCOMPLEX#1-#2, #5, #81RECOMBINANT
3Saccharomyces cerevisiae outer kinetochore Knl1c subcomplexCOMPLEXN-terminal truncation of Knl1(Spc105) residues 1-444#3-#41RECOMBINANT
4Saccharomyces cerevisiae outer kinetochore Ndc80cCOMPLEXAlso contains Saccharomyces cerevisiae kinetochore protein NDC80 (UniProtKB accession P40460), and Saccharomyces cerevisiae kinetochore protein NUF2 (UniProtKB accession P33895) with a C-terminal HRV-3C protease cleavage site and twin-strep-tag II affinity tag that were present in the imaged sample.#6-#71RECOMBINANT
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
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Crosslinked using glutaraldehyde with GraFix methodology. Crosslinker was quenched with 20 mM Tris pH 7.5.
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 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
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.66 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
4CTFFIND4.1CTF correction
7UCSF ChimeraXmodel fitting
8Cootmodel fitting
10RELION5initial Euler assignment
11RELION5final Euler assignment
13RELION53D reconstruction
14PHENIX1.21.2_5419:model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2592458
Details: Selected using Topaz particle picker trained on manually picked micrographs
3D reconstructionResolution: 7.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 18160 / Algorithm: BACK PROJECTION / Details: Refined using Blush regularization / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeChain-IDChain residue rangeInitial refinement model-IDPdb chain residue range
19S4Q

9s4q

Mt9S4QMt1-28911-289
29S4Q

9s4q

Nn9S4QNn1-20111-201
39S4Q

9s4q

Ds9S4QDs435-5761435-576
49S53

9s53

Ds9S53Ds229-3652229-365
59S4Q

9s4q

Ns9S4QNs97-216197-216
69S53

9s53

Ns9S53Ns2-9622-96
79S4Q

9s4q

Kl9S4QKl719-9171719-917
89S4Q

9s4q

Zw9S4QZw280-3851280-385
99S4Q

9s4q

249S4Q24143-2131143-213
109S4Q

9s4q

259S4Q25124-2211124-221
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01811404
ELECTRON MICROSCOPYf_angle_d0.44615354
ELECTRON MICROSCOPYf_dihedral_angle_d4.1821476
ELECTRON MICROSCOPYf_chiral_restr0.0361762
ELECTRON MICROSCOPYf_plane_restr0.0031969

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