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- EMDB-4581: Structure of inner kinetochore CCAN complex with mask1 -

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Basic information

Entry
Database: EMDB / ID: EMD-4581
TitleStructure of inner kinetochore CCAN complex with mask1
Map dataMap of CCAN with mask1
Sample
  • Complex: Inner kinetochore CCAN complex
    • Protein or peptide: Inner kinetochore subunit IML3
    • Protein or peptide: Inner kinetochore subunit CHL4
    • 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
Function / homology
Function and homology information


negative regulation of meiotic DNA double-strand break formation involved in reciprocal meiotic recombination / COMA complex / maintenance of meiotic sister chromatid cohesion / meiotic sister chromatid segregation / Mis6-Sim4 complex / centromere complex assembly / establishment of meiotic sister chromatid cohesion / ascospore formation / attachment of spindle microtubules to kinetochore / CENP-A containing chromatin assembly ...negative regulation of meiotic DNA double-strand break formation involved in reciprocal meiotic recombination / COMA complex / maintenance of meiotic sister chromatid cohesion / meiotic sister chromatid segregation / Mis6-Sim4 complex / centromere complex assembly / establishment of meiotic sister chromatid cohesion / ascospore formation / attachment of spindle microtubules to kinetochore / CENP-A containing chromatin assembly / outer kinetochore / protein localization to chromosome, centromeric region / establishment of mitotic sister chromatid cohesion / kinetochore assembly / spindle pole body / protein localization to kinetochore / mitotic spindle assembly checkpoint signaling / meiotic cell cycle / chromosome segregation / kinetochore / cell division / structural molecule activity / nucleus / cytoplasm
Similarity search - Function
Kinetochore subunit Nkp2/Cnl2 / Cnl2/NKP2 family protein / : / CENPU protein / Centromere protein O / Cenp-O kinetochore centromere component / Centromere protein Chl4/mis15/CENP-N / Kinetochore protein CHL4 like
Similarity search - Domain/homology
Inner kinetochore subunit IML3 / Inner kinetochore subunit AME1 / Inner kinetochore subunit CHL4 / 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: 3.45 Å
AuthorsYan K / Yang J / Zhang Z / McLaughlin SH / Chang L / Fasci D / Heck AJR / Barford D
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_UP_1201/6 United Kingdom
European CommissionINFRAIA project Epic-XS (Project 823839)
Cancer Research UKC576/A14109 United Kingdom
CitationJournal: Nature / Year: 2019
Title: Structure of the inner kinetochore CCAN complex assembled onto a centromeric nucleosome.
Authors: Kaige Yan / Jing Yang / Ziguo Zhang / Stephen H McLaughlin / Leifu Chang / Domenico Fasci / Ann E Ehrenhofer-Murray / Albert J R Heck / David Barford /
Abstract: In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes that, by assembling onto specialized ...In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes that, by assembling onto specialized Cenp-A nucleosomes, function to connect centromeric chromatin to microtubules of the mitotic spindle. Whereas the centromeres of vertebrate chromosomes comprise millions of DNA base pairs and attach to multiple microtubules, the simple point centromeres of budding yeast are connected to individual microtubules. All 16 budding yeast chromosomes assemble complete kinetochores using a single Cenp-A nucleosome (Cenp-A), each of which is perfectly centred on its cognate centromere. The inner and outer kinetochore modules are responsible for interacting with centromeric chromatin and microtubules, respectively. Here we describe the cryo-electron microscopy structure of the Saccharomyces cerevisiae inner kinetochore module, the constitutive centromere associated network (CCAN) complex, assembled onto a Cenp-A nucleosome (CCAN-Cenp-A). The structure explains the interdependency of the constituent subcomplexes of CCAN and shows how the Y-shaped opening of CCAN accommodates Cenp-A to enable specific CCAN subunits to contact the nucleosomal DNA and histone subunits. Interactions with the unwrapped DNA duplex at the two termini of Cenp-A are mediated predominantly by a DNA-binding groove in the Cenp-L-Cenp-N subcomplex. Disruption of these interactions impairs assembly of CCAN onto Cenp-A. Our data indicate a mechanism of Cenp-A nucleosome recognition by CCAN and how CCAN acts as a platform for assembly of the outer kinetochore to link centromeres to the mitotic spindle for chromosome segregation.
History
DepositionJan 31, 2019-
Header (metadata) releaseMar 6, 2019-
Map releaseOct 2, 2019-
UpdateNov 27, 2019-
Current statusNov 27, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.023
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.023
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6qlf
  • Surface level: 0.023
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4581.png

Downloads & links

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Map

FileDownload / File: emd_4581.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of CCAN with mask1
Voxel sizeX=Y=Z: 1.09 Å
Density
Contour LevelBy AUTHOR: 0.023 / Movie #1: 0.023
Minimum - Maximum-0.16614649 - 0.25293982
Average (Standard dev.)0.00014513815 (±0.0034088057)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 348.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.091.091.09
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z348.800348.800348.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.1660.2530.000

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Supplemental data

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Sample components

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Entire : Inner kinetochore CCAN complex

EntireName: Inner kinetochore CCAN complex
Components
  • Complex: Inner kinetochore CCAN complex
    • Protein or peptide: Inner kinetochore subunit IML3
    • Protein or peptide: Inner kinetochore subunit CHL4
    • 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: Inner kinetochore CCAN complex

SupramoleculeName: Inner kinetochore CCAN complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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Macromolecule #1: Inner kinetochore subunit IML3

MacromoleculeName: Inner kinetochore subunit IML3 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 28.093223 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MPYTWKFLGI SKQLSLENGI AKLNQLLNLE VDLDIQTIRV PSDPDGGTAA DEYIRYEMRL DISNLDEGTY SKFIFLGNSK MEVPMFLCY CGTDNRNEVV LQWLKAEYGV IMWPIKFEQK TMIKLADASI VHVTKENIEQ ITWFSSKLYF EPETQDKNLR Q FSIEIPRE ...String:
MPYTWKFLGI SKQLSLENGI AKLNQLLNLE VDLDIQTIRV PSDPDGGTAA DEYIRYEMRL DISNLDEGTY SKFIFLGNSK MEVPMFLCY CGTDNRNEVV LQWLKAEYGV IMWPIKFEQK TMIKLADASI VHVTKENIEQ ITWFSSKLYF EPETQDKNLR Q FSIEIPRE SCEGLALGYG NTMHPYNDAI VPYIYNETGM AVERLPLTSV ILAGHTKIMR ESIVTSTRSL RNRVLAVVLQ SI QFTSE

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Macromolecule #2: Inner kinetochore subunit CHL4

MacromoleculeName: Inner kinetochore subunit CHL4 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 53.538566 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSNELRLEDN YVPTSDTLVV FKQLMKLPVT VLYDLTLSWF AKFGGSFDGD IYLLTETLDL LIEKGVRRNV IVNRILYVYW PDGLNVFQL AEIDCHLMIS KPEKFKWLPS KALRGDGKPY VVKLQPAKFI ENLQTDLAKI YHCHVYMFKH PSLPVLITRI Q LFDSNNLF ...String:
MSNELRLEDN YVPTSDTLVV FKQLMKLPVT VLYDLTLSWF AKFGGSFDGD IYLLTETLDL LIEKGVRRNV IVNRILYVYW PDGLNVFQL AEIDCHLMIS KPEKFKWLPS KALRGDGKPY VVKLQPAKFI ENLQTDLAKI YHCHVYMFKH PSLPVLITRI Q LFDSNNLF LSTPNIGSIN KESLYNKLDK FQGKPLISRR PYYVAFPLNS PIIFHSVDKD IYARLVLQSI SRTISERETI IF KPVQKIP VKSIHNIMTL LGPSRFAESM GPWECYASAN FERSPLHDYK KHQGLTGKKV MVREFDDSFL NDDENFYGKE EPE IRRLRL EKNMIKFKGS ANGVMDQKYN DLKEFNEHVH NIRNGKKNED SGEPVYISRY SSLVPIEKVG FTLKNEINSR IITI KLKFN GNDIFGGLHE LCDKNLINID KVPGWLAGEN GSFSGTIMNG DFQREQVAKG GLLENLYFQ

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Macromolecule #3: Inner kinetochore subunit MCM21

MacromoleculeName: Inner kinetochore subunit MCM21 / type: protein_or_peptide / ID: 3 / 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

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Macromolecule #4: Inner kinetochore subunit CTF19

MacromoleculeName: Inner kinetochore subunit CTF19 / type: protein_or_peptide / ID: 4 / 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

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Macromolecule #5: Inner kinetochore subunit OKP1

MacromoleculeName: Inner kinetochore subunit OKP1 / type: protein_or_peptide / ID: 5 / 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

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Macromolecule #6: Inner kinetochore subunit AME1

MacromoleculeName: Inner kinetochore subunit AME1 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 37.081246 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

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Macromolecule #7: Inner kinetochore subunit NKP1

MacromoleculeName: Inner kinetochore subunit NKP1 / type: protein_or_peptide / ID: 7 / 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

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Macromolecule #8: Inner kinetochore subunit NKP2

MacromoleculeName: Inner kinetochore subunit NKP2 / type: protein_or_peptide / ID: 8 / 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

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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

Concentration1.0 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 32.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Reconstruction using SIMPLE_prime
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.45 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 465029

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