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- EMDB-21910: The yeast Ctf3 complex with Cnn1-Wip1 -

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Basic information

Entry
Database: EMDB / ID: EMD-21910
TitleThe yeast Ctf3 complex with Cnn1-Wip1
Map dataCMMCW
Sample
  • Complex: Single particle reconstruction of the Ctf3c bound to Cnn1-Wip1
    • Protein or peptide: Inner kinetochore subunit MCM16
    • Protein or peptide: Inner kinetochore subunit CTF3
    • Protein or peptide: Inner kinetochore subunit MCM22
    • Protein or peptide: Inner kinetochore subunit WIP1
    • Protein or peptide: Inner kinetochore subunit CNN1
Keywordskinetochore / cell cycle / chromosome segregation / CCAN
Function / homology
Function and homology information


negative regulation of kinetochore assembly / attachment of spindle microtubules to kinetochore / centromeric DNA binding / establishment of mitotic sister chromatid cohesion / mitotic spindle assembly checkpoint signaling / DNA replication initiation / meiotic cell cycle / chromosome segregation / kinetochore / cell division ...negative regulation of kinetochore assembly / attachment of spindle microtubules to kinetochore / centromeric DNA binding / establishment of mitotic sister chromatid cohesion / mitotic spindle assembly checkpoint signaling / DNA replication initiation / meiotic cell cycle / chromosome segregation / kinetochore / cell division / protein-containing complex binding / nucleus
Similarity search - Function
Inner kinetochore subunit CNN1 / Inner kinetochore subunit MCM22 / Inner kinetochore subunit MCM16 / Inner kinetochore subunit CTF3 / Inner kinetochore subunit WIP1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsHinshaw SM / Harrison SC
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Curr Biol / Year: 2020
Title: The Structural Basis for Kinetochore Stabilization by Cnn1/CENP-T.
Authors: Stephen M Hinshaw / Stephen C Harrison /
Abstract: Chromosome segregation depends on a regulated connection between spindle microtubules and centromeric DNA. The kinetochore mediates this connection and ensures it persists during anaphase, when ...Chromosome segregation depends on a regulated connection between spindle microtubules and centromeric DNA. The kinetochore mediates this connection and ensures it persists during anaphase, when sister chromatids must transit into daughter cells uninterrupted. The Ctf19 complex (Ctf19c) forms the centromeric base of the kinetochore in budding yeast. Biochemical experiments show that Ctf19c members associate hierarchically when purified from cell extract [1], an observation that is mostly explained by the structure of the complex [2]. The Ctf3 complex (Ctf3c), which is not required for the assembly of most other Ctf19c factors, disobeys the biochemical assembly hierarchy when observed in dividing cells that lack more basal components [3]. Thus, the biochemical experiments do not completely recapitulate the logic of centromeric Ctf19c assembly. We now present a high-resolution structure of the Ctf3c bound to the Cnn1-Wip1 heterodimer. Associated live-cell imaging experiments provide a mechanism for Ctf3c and Cnn1-Wip1 recruitment to the kinetochore. The mechanism suggests feedback regulation of Ctf19c assembly and unanticipated similarities in kinetochore organization between yeast and vertebrates.
History
DepositionMay 4, 2020-
Header (metadata) releaseJun 17, 2020-
Map releaseJun 17, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0235
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0235
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6wuc
  • Surface level: 0.0235
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21910.png

Downloads & links

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Map

FileDownload / File: emd_21910.map.gz / Format: CCP4 / Size: 93 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCMMCW
Voxel sizeX=Y=Z: 0.825 Å
Density
Contour LevelBy AUTHOR: 0.0235 / Movie #1: 0.0235
Minimum - Maximum-0.090992525 - 0.15769811
Average (Standard dev.)-0.0000039348643 (±0.0036952987)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions290290290
Spacing290290290
CellA=B=C: 239.25 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8250.8250.825
M x/y/z290290290
origin x/y/z0.0000.0000.000
length x/y/z239.250239.250239.250
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS290290290
D min/max/mean-0.0910.158-0.000

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Supplemental data

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Additional map: CMMCW-N

Fileemd_21910_additional.map
AnnotationCMMCW-N
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Single particle reconstruction of the Ctf3c bound to Cnn1-Wip1

EntireName: Single particle reconstruction of the Ctf3c bound to Cnn1-Wip1
Components
  • Complex: Single particle reconstruction of the Ctf3c bound to Cnn1-Wip1
    • Protein or peptide: Inner kinetochore subunit MCM16
    • Protein or peptide: Inner kinetochore subunit CTF3
    • Protein or peptide: Inner kinetochore subunit MCM22
    • Protein or peptide: Inner kinetochore subunit WIP1
    • Protein or peptide: Inner kinetochore subunit CNN1

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Supramolecule #1: Single particle reconstruction of the Ctf3c bound to Cnn1-Wip1

SupramoleculeName: Single particle reconstruction of the Ctf3c bound to Cnn1-Wip1
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 MCM16

MacromoleculeName: Inner kinetochore subunit MCM16 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 21.438359 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SNAMTNSSEK QWERIQQLEK EHVEVYRELL ITLDRLYLIR KHNHAVILSH TQQRLLEIRH QLQINLEKTA LLIRLLEKPD NTNVLFTKL QNLLEESNSL DYELLQSLGA QSSLHKQLIE SRAERDELMS KLIELSSKFP KPTIPPDDSD TAGKQVEVEK E NETIQELM IALQIHSGYT NISYTI

UniProtKB: Inner kinetochore subunit MCM16

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Macromolecule #2: Inner kinetochore subunit CTF3

MacromoleculeName: Inner kinetochore subunit CTF3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 84.617891 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SNAMSLILDD IILSLTNANE RTPPQALKTT LSLLYEKSKQ YGLSSPQLQA LVRLLCETSI IDTVTKVYIV ENCFLPDGYL TKELLLEII NHLGTPTVFS RYRIQTPPVL QSALCKWLVH VYFLFPVHSE REHNISSSIW LHLWQFSFLQ KWITPLVIWQ A TTPVDVKP ...String:
SNAMSLILDD IILSLTNANE RTPPQALKTT LSLLYEKSKQ YGLSSPQLQA LVRLLCETSI IDTVTKVYIV ENCFLPDGYL TKELLLEII NHLGTPTVFS RYRIQTPPVL QSALCKWLVH VYFLFPVHSE REHNISSSIW LHLWQFSFLQ KWITPLVIWQ A TTPVDVKP WKLSIIKRCA MHPGYRDAPG SATLILQRFQ CLVGASSQIT ESIITINCNR KTLKSHRNLK LDAHFLSILK RI LSRAHPA NFPADTVQNT IDMYLSEIHQ LGADSIYPLR LQSLPEYVPS DSTVSLWDVT SLEQLAQNWP QLHIPNDVDY MMK PSLNSN VLLPRKVMSR DSLKHLYSSI ILIKNSRDES SSPYEWCIWQ LKRCFAHQIE TPQEVIPIII SVSSMDNKLS SRII QTFCN LKYLKLDELT LKKVCGGILP LWKPELISGT REFFVKFMAS IFMWSTRDGH DNNCTFSETC FYVLQMITNW VLDDK LIAL GLTLLHDMQS LLTLDKIFNN ATSNRFSTMA FISSLDILTQ LSKQTKSDYA IQYLIVGPDI MNKVFSSDDP LLLSAA CRY LVATKNKLMQ YPSTNKFVRM QNQYIMDLTN YLYRNKVLSS KSLFGVSPDF FKQILENLYI PTADFKNAKF FTITGIP AL SYICIIILRR LETAENTKIK FTSGIINEET FNNFFRVHHD EIGQHGWIKG VNNIHDLRVK ILMHLSNTAN PYRDIAAF L FTYLKSLSKY SVQNS

UniProtKB: Inner kinetochore subunit CTF3

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Macromolecule #3: Inner kinetochore subunit MCM22

MacromoleculeName: Inner kinetochore subunit MCM22 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 27.874799 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SNAMDVEKDV LDVYIKNLEN QIGNKRYFLK QAQGAIDEIT KRSLDTEGKP VNSEVFTELL RKPMFFSERA DPIGFSLTSN FLSLRAQSS SEWLSLMNDQ SVDQKAMLLL QNNINSDLKE LLRKLQHQMT IMDSKKQDHA HIRTRKARNK ELWDSLADFL K GYLVPNLD ...String:
SNAMDVEKDV LDVYIKNLEN QIGNKRYFLK QAQGAIDEIT KRSLDTEGKP VNSEVFTELL RKPMFFSERA DPIGFSLTSN FLSLRAQSS SEWLSLMNDQ SVDQKAMLLL QNNINSDLKE LLRKLQHQMT IMDSKKQDHA HIRTRKARNK ELWDSLADFL K GYLVPNLD DNDESIDSLT NEVMLLMKRL IEHDLNLTLN DFSSKTIPIY RLLLRANIIT VIEGSTNPGT KYIKLIDFNE TS LT

UniProtKB: Inner kinetochore subunit MCM22

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Macromolecule #4: Inner kinetochore subunit WIP1

MacromoleculeName: Inner kinetochore subunit WIP1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 10.527717 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
SNAMDTEALA NYLLRQLSLD AEENKLEDLL QRQNEDQESS QEYNKKLLLA CGFQAILRKI LLDARTRATA EGLREVYPYH IEAATQAFL DSQ

UniProtKB: Inner kinetochore subunit WIP1

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Macromolecule #5: Inner kinetochore subunit CNN1

MacromoleculeName: Inner kinetochore subunit CNN1 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 41.359785 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSTPRKAAGN NENTEVSEIR TPFRERALEE QRLKDEVLIR NTPGYRKLLS ASTKSHDILN KDPNEVRSFL QDLSQVLARK SQGNDTTTN KTQARNLIDE LAYEESQPEE NELLRSRSEK LTDNNIGNET QPDYTSLSQT VFAKLQERDK GLKSRKIDPI I IQDVPTTG ...String:
MSTPRKAAGN NENTEVSEIR TPFRERALEE QRLKDEVLIR NTPGYRKLLS ASTKSHDILN KDPNEVRSFL QDLSQVLARK SQGNDTTTN KTQARNLIDE LAYEESQPEE NELLRSRSEK LTDNNIGNET QPDYTSLSQT VFAKLQERDK GLKSRKIDPI I IQDVPTTG HEDELTVHSP DKANSISMEV LRTSPSIGMD QVDEPPVRDP VPISITQQEE PLSEDLPSDD KEETEEAENE DY SFENTSD ENLDDIGNDP IRLNVPAVRR SSIKPLQIMD LKHLTRQFLN ENRIILPKQT WSTIQEESLN IMDFLKQKIG TLQ KQELVD SFIDMGIINN VDDMFELAHE LLPLELQSRI ESYLF

UniProtKB: Inner kinetochore subunit CNN1

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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: 8.5
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: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 109996
FSC plot (resolution estimation)

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