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- EMDB-17225: Cryo-EM structure of yeast CENP-OPQU+ bound to the CENP-A N-terminus -

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Basic information

Entry
Database: EMDB / ID: EMD-17225
TitleCryo-EM structure of yeast CENP-OPQU+ bound to the CENP-A N-terminus
Map data
Sample
  • Complex: A complex of CBF1-CCAN bound to centromeric C0N3 DNA
    • Protein or peptide: Inner kinetochore subunit MCM21
    • Protein or peptide: Inner kinetochore subunit CTF19
    • Protein or peptide: Inner kinetochore subunit OKP1
    • Protein or peptide: Inner kinetochore subunit AME1
    • Protein or peptide: Inner kinetochore subunit NKP1
    • Protein or peptide: Inner kinetochore subunit NKP2
Keywordskinetochore / point centromere / CENP-A nucleosome / topological entrapment / centromeric DNA / CELL CYCLE
Function / homology
Function and homology information


negative regulation of meiotic DNA double-strand break formation involved in reciprocal meiotic recombination / COMA complex / Mis6-Sim4 complex / centromere complex assembly / attachment of spindle microtubules to kinetochore / protein localization to chromosome, centromeric region / establishment of mitotic sister chromatid cohesion / protein localization to kinetochore / spindle pole body / mitotic spindle assembly checkpoint signaling ...negative regulation of meiotic DNA double-strand break formation involved in reciprocal meiotic recombination / COMA complex / Mis6-Sim4 complex / centromere complex assembly / attachment of spindle microtubules to kinetochore / protein localization to chromosome, centromeric region / establishment of mitotic sister chromatid cohesion / protein localization to kinetochore / spindle pole body / mitotic spindle assembly checkpoint signaling / meiotic cell cycle / chromosome segregation / kinetochore / cell division / nucleus / cytoplasm
Similarity search - Function
Kinetochore subunit Nkp2/Cnl2 / Cnl2/NKP2 family protein / : / CENPU protein / Centromere protein O / Cenp-O kinetochore centromere component
Similarity search - Domain/homology
Inner kinetochore subunit AME1 / Inner kinetochore subunit OKP1 / Inner kinetochore subunit CTF19 / Inner kinetochore subunit NKP2 / Inner kinetochore subunit MCM21 / Inner kinetochore subunit NKP1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsDendooven TD / Zhang Z / Yang J / McLaughlin S / Schwabb J / Scheres S / Yatskevich S
Funding support United Kingdom, Germany, 4 items
OrganizationGrant numberCountry
UK Research and Innovation (UKRI)MC_UP_1201/6 United Kingdom
Cancer Research UKC576/A14109 United Kingdom
UK Research and Innovation (UKRI)MC_UP_ A025_1013 United Kingdom
Boehringer Ingelheim Fonds (BIF) Germany
CitationJournal: Sci Adv / Year: 2023
Title: Cryo-EM structure of the complete inner kinetochore of the budding yeast point centromere.
Authors: Tom Dendooven / Ziguo Zhang / Jing Yang / Stephen H McLaughlin / Johannes Schwab / Sjors H W Scheres / Stanislau Yatskevich / David Barford /
Abstract: The point centromere of budding yeast specifies assembly of the large kinetochore complex to mediate chromatid segregation. Kinetochores comprise the centromere-associated inner kinetochore (CCAN) ...The point centromere of budding yeast specifies assembly of the large kinetochore complex to mediate chromatid segregation. Kinetochores comprise the centromere-associated inner kinetochore (CCAN) complex and the microtubule-binding outer kinetochore KNL1-MIS12-NDC80 (KMN) network. The budding yeast inner kinetochore also contains the DNA binding centromere-binding factor 1 (CBF1) and CBF3 complexes. We determined the cryo-electron microscopy structure of the yeast inner kinetochore assembled onto the centromere-specific centromere protein A nucleosomes (CENP-A). This revealed a central CENP-A with extensively unwrapped DNA ends. These free DNA duplexes bind two CCAN protomers, one of which entraps DNA topologically, positioned on the centromere DNA element I (CDEI) motif by CBF1. The two CCAN protomers are linked through CBF3 forming an arch-like configuration. With a structural mechanism for how CENP-A can also be linked to KMN involving only CENP-QU, we present a model for inner kinetochore assembly onto a point centromere and how it organizes the outer kinetochore for chromosome attachment to the mitotic spindle.
History
DepositionApr 26, 2023-
Header (metadata) releaseAug 9, 2023-
Map releaseAug 9, 2023-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17225.png

Downloads & links

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Map

FileDownload / File: emd_17225.map.gz / Format: CCP4 / Size: 18.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.65 Å/pix.
x 170 pix.
= 280.84 Å		1.65 Å/pix.
x 170 pix.
= 280.84 Å		1.65 Å/pix.
x 170 pix.
= 280.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.652 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.679
Minimum - Maximum-1.6564386 - 3.792524
Average (Standard dev.)0.0005520734 (±0.061818946)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions170170170
Spacing170170170
CellA=B=C: 280.84 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_17225_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_17225_half_map_2.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 : A complex of CBF1-CCAN bound to centromeric C0N3 DNA

EntireName: A complex of CBF1-CCAN bound to centromeric C0N3 DNA
Components
  • Complex: A complex of CBF1-CCAN bound to centromeric C0N3 DNA
    • Protein or peptide: Inner kinetochore subunit MCM21
    • Protein or peptide: Inner kinetochore subunit CTF19
    • Protein or peptide: Inner kinetochore subunit OKP1
    • Protein or peptide: Inner kinetochore subunit AME1
    • Protein or peptide: Inner kinetochore subunit NKP1
    • Protein or peptide: Inner kinetochore subunit NKP2

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Supramolecule #1: A complex of CBF1-CCAN bound to centromeric C0N3 DNA

SupramoleculeName: A complex of CBF1-CCAN bound to centromeric C0N3 DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: Inner kinetochore subunit MCM21

MacromoleculeName: Inner kinetochore subunit MCM21 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 43.028879 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSRIDDLQQD IESLLSEINS LEESREKLKA KIKDKRKNEE SANPIVQEFE DLFDQFPQLN NFLFNEHPEL EETDDKDISR AQADIPATP IPYEPKKRAK LENEEILPEQ EWVLKTQPMV QHQMFDPGVA DLLDTDILTS PSKRKRKLKI DDISTSDRSE L EDYIVLEN ...String:
MSRIDDLQQD IESLLSEINS LEESREKLKA KIKDKRKNEE SANPIVQEFE DLFDQFPQLN NFLFNEHPEL EETDDKDISR AQADIPATP IPYEPKKRAK LENEEILPEQ EWVLKTQPMV QHQMFDPGVA DLLDTDILTS PSKRKRKLKI DDISTSDRSE L EDYIVLEN VYRMFGITFF PLVDPIDLKI KDASGEIFVD REMLGIRLEV FSERTSQFEK PHYVLLKKRI KSNSWFLFKH TI PSFIDVQ GIFDDTNGGL VISHDDAYLF AKRVFLQLVE VQKRRQIFKD LEAKKIIHDL DLDLESSMVS FFVKDIKVEL FVK QNEIVS CSILDDIHDF SQNNKSKWEI ALLGSLDDLE LKLNHSFATI FK

UniProtKB: Inner kinetochore subunit MCM21

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Macromolecule #2: Inner kinetochore subunit CTF19

MacromoleculeName: Inner kinetochore subunit CTF19 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 42.841113 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MDFTSDTTNS HDTSNSHLSL EDAVGTHHAG EADVNIDGDE KQQLSLLDDD QVRALKLQEE KDALLTRRNT LLQEIQTYQN ILMKENNSK TKNGDILQND ITQDFLNLIS ISSSNPNSAI SDRKRVERIN GLTNLQKELV TKYDTLPLLN MNLRLSYLRD H TYPHLQVS ...String:
MDFTSDTTNS HDTSNSHLSL EDAVGTHHAG EADVNIDGDE KQQLSLLDDD QVRALKLQEE KDALLTRRNT LLQEIQTYQN ILMKENNSK TKNGDILQND ITQDFLNLIS ISSSNPNSAI SDRKRVERIN GLTNLQKELV TKYDTLPLLN MNLRLSYLRD H TYPHLQVS VQSRDRVHND GIEVLVVNYK FCRNTMNPFE IQFKMFYKFE DSTLLKWEIL RISTNVRLKA KQLLATRNFQ KC LLSLYEF DKIKSKKTGI FQNLINLLKR KTRCYLMNNS DSLIVERVIR EGRLTTIKLQ INFIITMPGE RGKPRNCFLP MSK ISIALW KGGERFNQID LDEICYGLIK EYGVKTGLKE ICNVCLFPDM YAR

UniProtKB: Inner kinetochore subunit CTF19

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Macromolecule #3: Inner kinetochore subunit OKP1

MacromoleculeName: Inner kinetochore subunit OKP1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 47.427246 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MAADRDNFLQ NIENDSINNG QAMDLSPNRS SSESDSSILM NVNDIKTLRL DVAPEAKSTQ SKKSLFYENS DDAEEGEIEE RTNKEEGQY HHKGSKQLRF EVGKESTGKL QSHLSDGSAT SGEGNVRPWE FRKVIQAEYR ERLPRNYELK HWKKPSKIMI G SILRLLET ...String:
MAADRDNFLQ NIENDSINNG QAMDLSPNRS SSESDSSILM NVNDIKTLRL DVAPEAKSTQ SKKSLFYENS DDAEEGEIEE RTNKEEGQY HHKGSKQLRF EVGKESTGKL QSHLSDGSAT SGEGNVRPWE FRKVIQAEYR ERLPRNYELK HWKKPSKIMI G SILRLLET NTVSALDSVF EKYEKEMNQM THGDNNEVKR IYSKKERLLE IILTKIKKKL RQAKFPSRIS ERDLDIEYIY SK RQFIQNR YSQELQNNER LEAILSREQN LLEETRKLCM NLKTNNKKRL TEKLIQKDLH PVLNKAMEYT YGLESTNGFM HPD GPVTFR NDSHELNLML NDPIKSTADV RLDKEEVLSL LPSLKEYTKK SKELKETMGQ MISDSHEEEI KEVFVPHHES HQDK TEEDI H

UniProtKB: Inner kinetochore subunit OKP1

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Macromolecule #4: Inner kinetochore subunit AME1

MacromoleculeName: Inner kinetochore subunit AME1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 37.506723 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MDRDTKLAFR LRGSHSRRTD DIDDDVIVFK TPNAVYREEN SPIQSPVQPI LSSPKLANSF EFPITTNNVN AQDRHEHGYQ PLDAEDYPM IDSENKSLIS ESPQNVRNDE DLTTRYNFDD IPIRQLSSSI TSVTTIDVLS SLFINLFEND LIPQALKDFN K SDDDQFRK ...String:
MDRDTKLAFR LRGSHSRRTD DIDDDVIVFK TPNAVYREEN SPIQSPVQPI LSSPKLANSF EFPITTNNVN AQDRHEHGYQ PLDAEDYPM IDSENKSLIS ESPQNVRNDE DLTTRYNFDD IPIRQLSSSI TSVTTIDVLS SLFINLFEND LIPQALKDFN K SDDDQFRK LLYKLDLRLF QTISDQMTRD LKDILDINVS NNELCYQLKQ VLARKEDLNQ QIISVRNEIQ ELKAGKDWHD LQ NEQAKLN DKVKLNKRLN DLTSTLLGKY EGDRKIMSQD SEDDSIRDDS NILDIAHFVD LMDPYNGLLK KINKINENLS NEL QPSL

UniProtKB: Inner kinetochore subunit AME1

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Macromolecule #5: Inner kinetochore subunit NKP1

MacromoleculeName: Inner kinetochore subunit NKP1 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 27.006451 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MTDTYNSISN FIENELTALL SSDDYLMDDL AGELPNEVCR LLKAQVIEKR KDAMSRGKQD LLSKEIYDNE SELRASQSQQ IMELVGDIP KYSLGSELRN RVEGEPQSTS IERLIEDVLK LPQMEVADEE EVEVENDLKV LSEYSNLRKD LILKCQALQI G ESKLSDIL ...String:
MTDTYNSISN FIENELTALL SSDDYLMDDL AGELPNEVCR LLKAQVIEKR KDAMSRGKQD LLSKEIYDNE SELRASQSQQ IMELVGDIP KYSLGSELRN RVEGEPQSTS IERLIEDVLK LPQMEVADEE EVEVENDLKV LSEYSNLRKD LILKCQALQI G ESKLSDIL SQTNSINSLT TSIKEASEDD DISEYFATYN GKLVVALEEM KLLLEEAVKT FGNSPEKREK IKKILSELKK

UniProtKB: Inner kinetochore subunit NKP1

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Macromolecule #6: Inner kinetochore subunit NKP2

MacromoleculeName: Inner kinetochore subunit NKP2 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 17.877033 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MNSEQLLHNY VSDSLLTTLI SFQEFKQQLQ SYTSDEQQLQ HWYELLQARD ARVTSELEAR IKQFFITLRS RLLRFLESEQ LSHSLSLET LIDALYKIND LLQQRLQILD DAIQEKTSEL AEFENMVRSP SAGDNAIPGL LQIIQSYINL LEEN

UniProtKB: Inner kinetochore subunit NKP2

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 595147
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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