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- PDB-7qoo: Structure of the human inner kinetochore CCAN complex -

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Basic information

Entry
Database: PDB / ID: 7qoo
TitleStructure of the human inner kinetochore CCAN complex
Components
  • (Centromere protein ...) x 14
  • Unknown protein
KeywordsCELL CYCLE / INNER KINETOCHORE / CCAN / COMPLEX / DNA BINDING PROTEIN
Function / homology
Function and homology information


positive regulation of protein localization to kinetochore / Mis6-Sim4 complex / centromere complex assembly / kinetochore organization / metaphase chromosome alignment / spindle attachment to meiosis I kinetochore / inner kinetochore / kinetochore binding / centromeric DNA binding / sex differentiation ...positive regulation of protein localization to kinetochore / Mis6-Sim4 complex / centromere complex assembly / kinetochore organization / metaphase chromosome alignment / spindle attachment to meiosis I kinetochore / inner kinetochore / kinetochore binding / centromeric DNA binding / sex differentiation / CENP-A containing chromatin assembly / chordate embryonic development / negative regulation of epithelial cell apoptotic process / attachment of mitotic spindle microtubules to kinetochore / kinetochore assembly / condensed chromosome, centromeric region / mitotic sister chromatid segregation / chromosome, centromeric region / centriolar satellite / chromosome organization / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / pericentric heterochromatin / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Deposition of new CENPA-containing nucleosomes at the centromere / Resolution of Sister Chromatid Cohesion / NRIF signals cell death from the nucleus / mitotic spindle organization / chromosome segregation / positive regulation of epithelial cell proliferation / RHO GTPases Activate Formins / kinetochore / nuclear matrix / Separation of Sister Chromatids / actin cytoskeleton / mitotic cell cycle / chromosome / midbody / nuclear body / cell adhesion / protein heterodimerization activity / cell division / apoptotic process / nucleolus / regulation of DNA-templated transcription / signal transduction / DNA binding / nucleoplasm / membrane / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Centromere protein W / CENP-W protein / Centromere protein T / Centromere kinetochore component CENP-T, N-terminal domain / Centromere kinetochore component CENP-T N-terminus / Centromere subunit L / Kinetochore complex Sim4 subunit Fta1 / Centromere protein R / Centromere protein Q / Centromere protein U ...Centromere protein W / CENP-W protein / Centromere protein T / Centromere kinetochore component CENP-T, N-terminal domain / Centromere kinetochore component CENP-T N-terminus / Centromere subunit L / Kinetochore complex Sim4 subunit Fta1 / Centromere protein R / Centromere protein Q / Centromere protein U / Centromere protein P / Centromere protein H / Kinetochore component, CENP-R / CENP-Q, a CENPA-CAD centromere complex subunit / CENP-A-nucleosome distal (CAD) centromere subunit, CENP-P / CENP-A nucleosome associated complex (NAC) subunit / Centromere protein H, C-terminal / Centromere protein Cenp-M / Centromere protein Cenp-K / Centromere protein H (CENP-H) / Centromere protein M (CENP-M) / Centromere-associated protein K / Centromere protein I / Mis6 / CENP-C, middle DNMT3B-binding domain / Centromere assembly component CENP-C middle DNMT3B-binding region / Centromere protein O / Cenp-O kinetochore centromere component / Kinetochore assembly subunit CENP-C, N-terminal domain / Kinetochore assembly subunit CENP-C N-terminal / Mif2/CENP-C cupin domain / Centromere protein C/Mif2/cnp3 / Mif2/CENP-C like / Centromere protein Chl4/mis15/CENP-N / Kinetochore protein CHL4 like / RmlC-like cupin domain superfamily / RmlC-like jelly roll fold / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / Histone-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Centromere protein C / Centromere protein R / Centromere protein W / Centromere protein P / Centromere protein U / Centromere protein Q / Centromere protein L / Centromere protein I / Centromere protein T / Centromere protein N ...Centromere protein C / Centromere protein R / Centromere protein W / Centromere protein P / Centromere protein U / Centromere protein Q / Centromere protein L / Centromere protein I / Centromere protein T / Centromere protein N / Centromere protein K / Centromere protein O / Centromere protein H / Centromere protein M
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsVetter, I.R. / Pesenti, M. / Raisch, T.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Mol Cell / Year: 2022
Title: Structure of the human inner kinetochore CCAN complex and its significance for human centromere organization.
Authors: Marion E Pesenti / Tobias Raisch / Duccio Conti / Kai Walstein / Ingrid Hoffmann / Dorothee Vogt / Daniel Prumbaum / Ingrid R Vetter / Stefan Raunser / Andrea Musacchio /
Abstract: Centromeres are specialized chromosome loci that seed the kinetochore, a large protein complex that effects chromosome segregation. A 16-subunit complex, the constitutive centromere associated ...Centromeres are specialized chromosome loci that seed the kinetochore, a large protein complex that effects chromosome segregation. A 16-subunit complex, the constitutive centromere associated network (CCAN), connects between the specialized centromeric chromatin, marked by the histone H3 variant CENP-A, and the spindle-binding moiety of the kinetochore. Here, we report a cryo-electron microscopy structure of human CCAN. We highlight unique features such as the pseudo GTPase CENP-M and report how a crucial CENP-C motif binds the CENP-LN complex. The CCAN structure has implications for the mechanism of specific recognition of the CENP-A nucleosome. A model consistent with our structure depicts the CENP-C-bound nucleosome as connected to the CCAN through extended, flexible regions of CENP-C. An alternative model identifies both CENP-C and CENP-N as specificity determinants but requires CENP-N to bind CENP-A in a mode distinct from the classical nucleosome octamer.
History
DepositionDec 24, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 8, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 15, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: Centromere protein C
H: Centromere protein H
I: Centromere protein I
K: Centromere protein K
L: Centromere protein L
M: Centromere protein M
N: Centromere protein N
O: Centromere protein O
P: Centromere protein P
U: Centromere protein U
Q: Centromere protein Q
R: Centromere protein R
T: Centromere protein T
W: Centromere protein W
X: Unknown protein


Theoretical massNumber of molelcules
Total (without water)544,71715
Polymers544,71715
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Centromere protein ... , 14 types, 14 molecules CHIKLMNOPUQRTW

#1: Protein Centromere protein C / / CENP-C / Centromere autoantigen C / Centromere protein C 1 / CENP-C 1 / Interphase centromere ...CENP-C / Centromere autoantigen C / Centromere protein C 1 / CENP-C 1 / Interphase centromere complex protein 7


Mass: 61856.004 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CENPC, CENPC1, ICEN7 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q03188
#2: Protein Centromere protein H / / CENP-H / Interphase centromere complex protein 35


Mass: 28520.941 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CENPH, ICEN35 / Production host: Trichoplusia ni (cabbage looper) / Strain (production host): Tnao38 / References: UniProt: Q9H3R5
#3: Protein Centromere protein I / / CENP-I / FSH primary response protein 1 / Follicle-stimulating hormone primary response protein / ...CENP-I / FSH primary response protein 1 / Follicle-stimulating hormone primary response protein / Interphase centromere complex protein 19 / Leucine-rich primary response protein 1


Mass: 86820.188 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CENPI, FSHPRH1, ICEN19, LRPR1 / Production host: Trichoplusia ni (cabbage looper) / Strain (production host): Tnao38 / References: UniProt: Q92674
#4: Protein Centromere protein K / / CENP-K / Interphase centromere complex protein 37 / Protein AF-5alpha / p33


Mass: 31696.070 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CENPK, ICEN37, FKSG14 / Production host: Trichoplusia ni (cabbage looper) / Strain (production host): Tnao38 / References: UniProt: Q9BS16
#5: Protein Centromere protein L / / CENP-L / Interphase centromere complex protein 33


Mass: 39039.641 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CENPL, C1orf155, ICEN33 / Production host: Trichoplusia ni (cabbage looper) / Strain (production host): Tnao38 / References: UniProt: Q8N0S6
#6: Protein Centromere protein M / CENPM / CENP-M / Interphase centromere complex protein 39 / Proliferation-associated nuclear element protein 1


Mass: 19761.945 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CENPM, C22orf18, ICEN39, PANE1 / Production host: Trichoplusia ni (cabbage looper) / Strain (production host): Tnao38 / References: UniProt: Q9NSP4
#7: Protein Centromere protein N / / CENP-N / Interphase centromere complex protein 32


Mass: 39609.551 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CENPN, C16orf60, ICEN32, BM-309 / Production host: Trichoplusia ni (cabbage looper) / Strain (production host): Tnao38 / References: UniProt: Q96H22
#8: Protein Centromere protein O / CENPO / CENP-O / Interphase centromere complex protein 36


Mass: 33830.637 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CENPO, ICEN36, MCM21R / Production host: Trichoplusia ni (cabbage looper) / Strain (production host): Tnao38 / References: UniProt: Q9BU64
#9: Protein Centromere protein P / / CENP-P


Mass: 33210.949 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CENPP / Production host: Trichoplusia ni (cabbage looper) / Strain (production host): Tnao38 / References: UniProt: Q6IPU0
#10: Protein Centromere protein U / / CENP-U / Centromere protein of 50 kDa / CENP-50 / Interphase centromere complex protein 24 / KSHV ...CENP-U / Centromere protein of 50 kDa / CENP-50 / Interphase centromere complex protein 24 / KSHV latent nuclear antigen-interacting protein 1 / MLF1-interacting protein / Polo-box-interacting protein 1


Mass: 47609.766 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CENPU, ICEN24, KLIP1, MLF1IP, PBIP1 / Production host: Trichoplusia ni (cabbage looper) / Strain (production host): Tnao38 / References: UniProt: Q71F23
#11: Protein Centromere protein Q / / CENP-Q


Mass: 30648.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CENPQ, C6orf139 / Production host: Trichoplusia ni (cabbage looper) / Strain (production host): Tnao38 / References: UniProt: Q7L2Z9
#12: Protein Centromere protein R / / CENP-R / Beta-3-endonexin / Integrin beta-3-binding protein / Nuclear receptor-interacting factor 3


Mass: 20228.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ITGB3BP, CENPR, NRIF3 / Production host: Trichoplusia ni (cabbage looper) / Strain (production host): Tnao38 / References: UniProt: Q13352
#13: Protein Centromere protein T / / CENP-T / Interphase centromere complex protein 22


Mass: 60502.613 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CENPT, C16orf56, ICEN22 / Plasmid: pETDuet / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): -Codon-plus-RIL / References: UniProt: Q96BT3
#14: Protein Centromere protein W / / CENP-W / Cancer-up-regulated gene 2 protein


Mass: 10087.236 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CENPW, C6orf173, CUG2 / Plasmid: pETDuet / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): -Codon-plus-RIL / References: UniProt: Q5EE01

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Protein/peptide , 1 types, 1 molecules X

#15: Protein/peptide Unknown protein


Mass: 1294.587 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Trichoplusia ni (cabbage looper) / Strain (production host): Tnao38

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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

ComponentName: human inner kinetochore CCAN complex / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.451 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper) / Strain: Tnao38
Buffer solutionpH: 6.8 / Details: 0.0025% Triton
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 286 K / Details: blot for 3.5 seconds at blot force -3

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 1200 nm / Nominal defocus min: 600 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 76.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 1540
Details: Images were collected in movie mode with 80 frames per image in superresolution mode with 0.35 A/px (0.7A native)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV / Phase plate: VOLTA PHASE PLATE

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Processing

EM software
IDNameVersionCategory
1crYOLOparticle selection
2EPUimage acquisition
4CTFFINDCTF correction
7RELION3.1.2model fitting
9RELION3.1.2model refinement
12RELION3.1.2classification
13RELION3.1.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 140910
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 25206 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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