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- PDB-6muo: CENP-A nucleosome bound by two copies of CENP-C(CD) and one copy ... -

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Entry
Database: PDB / ID: 6muo
TitleCENP-A nucleosome bound by two copies of CENP-C(CD) and one copy CENP-N(NT)
Components
  • (Centromere protein ...) x 2
  • (DNA/RNA (147- ...) x 2
  • Histone H2A type 1-C
  • Histone H2B type 2-F
  • Histone H3-like centromeric protein A
  • Histone H4
KeywordsNUCLEAR PROTEIN / centromere / CENP-A / kinetochore / nucleosome
Function / homology
Function and homology information


monopolar spindle attachment to meiosis I kinetochore / inner kinetochore / centromeric DNA binding / attachment of mitotic spindle microtubules to kinetochore / kinetochore assembly / condensed chromosome, centromeric region / protein localization to chromosome, centromeric region / establishment of mitotic spindle orientation / negative regulation of megakaryocyte differentiation / Packaging Of Telomere Ends ...monopolar spindle attachment to meiosis I kinetochore / inner kinetochore / centromeric DNA binding / attachment of mitotic spindle microtubules to kinetochore / kinetochore assembly / condensed chromosome, centromeric region / protein localization to chromosome, centromeric region / establishment of mitotic spindle orientation / negative regulation of megakaryocyte differentiation / Packaging Of Telomere Ends / chromosome, centromeric region / Cleavage of the damaged purine / Recognition and association of DNA glycosylase with site containing an affected purine / Deposition of new CENPA-containing nucleosomes at the centromere / mitotic cytokinesis / DNA replication-independent chromatin assembly / Cleavage of the damaged pyrimidine / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Inhibition of DNA recombination at telomere / Meiotic synapsis / pericentric heterochromatin / heterochromatin assembly => GO:0031507 / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / telomere organization / RNA Polymerase I Promoter Opening / SUMOylation of chromatin organization proteins / DNA replication-dependent chromatin assembly / Resolution of Sister Chromatid Cohesion / DNA methylation / HCMV Late Events / SIRT1 negatively regulates rRNA expression / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / Condensation of Prophase Chromosomes / PRC2 methylates histones and DNA / Defective pyroptosis / RNA Polymerase I Promoter Escape / HDACs deacetylate histones / nuclear chromosome / RHO GTPases Activate Formins / Transcriptional regulation by small RNAs / NoRC negatively regulates rRNA expression / Nonhomologous End-Joining (NHEJ) / kinetochore / Formation of the beta-catenin:TCF transactivating complex / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / B-WICH complex positively regulates rRNA expression / chromosome segregation / DNA-templated transcription, initiation / Metalloprotease DUBs / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / DNA Damage/Telomere Stress Induced Senescence / G2/M DNA damage checkpoint / RMTs methylate histone arginines / nucleosome assembly / HCMV Early Events / HDMs demethylate histones / Pre-NOTCH Transcription and Translation / Meiotic recombination / PKMTs methylate histone lysines / nucleosome / Activation of anterior HOX genes in hindbrain development during early embryogenesis / UCH proteinases / Transcriptional regulation of granulopoiesis / Separation of Sister Chromatids / midbody / RUNX1 regulates transcription of genes involved in differentiation of HSCs / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / Processing of DNA double-strand break ends / mitotic cell cycle / HATs acetylate histones / Senescence-Associated Secretory Phenotype (SASP) / chromosome, telomeric region / Oxidative Stress Induced Senescence / Estrogen-dependent gene expression / nuclear body / Ub-specific processing proteases / protein heterodimerization activity / cell division / Amyloid fiber formation / negative regulation of cell population proliferation / protein domain specific binding / chromatin binding / protein-containing complex / RNA binding / DNA binding / extracellular exosome / membrane / extracellular region / nucleoplasm / identical protein binding / nucleus / cytosol
Similarity search - Function
Centromere assembly component CENP-C middle DNMT3B-binding region / CENP-C, middle DNMT3B-binding domain / Kinetochore assembly subunit CENP-C, N-terminal domain / Kinetochore assembly subunit CENP-C N-terminal / Centromere protein Chl4/mis15/CENP-N / Kinetochore protein CHL4 like / Centromere protein C/Mif2/cnp3 / Mif2/CENP-C cupin domain / Mif2/CENP-C like / Histone, subunit A ...Centromere assembly component CENP-C middle DNMT3B-binding region / CENP-C, middle DNMT3B-binding domain / Kinetochore assembly subunit CENP-C, N-terminal domain / Kinetochore assembly subunit CENP-C N-terminal / Centromere protein Chl4/mis15/CENP-N / Kinetochore protein CHL4 like / Centromere protein C/Mif2/cnp3 / Mif2/CENP-C cupin domain / Mif2/CENP-C like / Histone, subunit A / Histone, subunit A / RmlC-like cupin domain superfamily / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone 2A / Histone H2A / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / Centromere kinetochore component CENP-T histone fold / CENP-T/Histone H4, histone fold / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF) / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / RmlC-like jelly roll fold / Core histone H2A/H2B/H3/H4 / Histone H2A/H2B/H3 / Histone-fold / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
Histone H2B type 2-F / Histone H2A type 1-C / Centromere protein C / DNA / Histone H4 / Histone H3-like centromeric protein A / DNA (> 100) / DNA (> 10) / Centromere protein N
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsAllu, P.K. / Black, B.E.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)GM123233 GM130302 United States
CitationJournal: Curr Biol / Year: 2019
Title: Structure of the Human Core Centromeric Nucleosome Complex.
Authors: Praveen Kumar Allu / Jennine M Dawicki-McKenna / Trevor Van Eeuwen / Moriya Slavin / Merav Braitbard / Chen Xu / Nir Kalisman / Kenji Murakami / Ben E Black /
Abstract: Centromeric nucleosomes are at the interface of the chromosome and the kinetochore that connects to spindle microtubules in mitosis. The core centromeric nucleosome complex (CCNC) harbors the ...Centromeric nucleosomes are at the interface of the chromosome and the kinetochore that connects to spindle microtubules in mitosis. The core centromeric nucleosome complex (CCNC) harbors the histone H3 variant, CENP-A, and its binding proteins, CENP-C (through its central domain; CD) and CENP-N (through its N-terminal domain; NT). CENP-C can engage nucleosomes through two domains: the CD and the CENP-C motif (CM). CENP-C is part of the CCNC by virtue of its high specificity for CENP-A nucleosomes and ability to stabilize CENP-A at the centromere. CENP-C is thought to engage a neighboring nucleosome, either one containing conventional H3 or CENP-A, and a crystal structure of a nucleosome complex containing two copies of CENP-C was reported. Recent structures containing a single copy of CENP-N bound to the CENP-A nucleosome in the absence of CENP-C were reported. Here, we find that one copy of CENP-N is lost for every two copies of CENP-C on centromeric chromatin just prior to kinetochore formation. We present the structures of symmetric and asymmetric forms of the CCNC that vary in CENP-N stoichiometry. Our structures explain how the central domain of CENP-C achieves its high specificity for CENP-A nucleosomes and how CENP-C and CENP-N sandwich the histone H4 tail. The natural centromeric DNA path in our structures corresponds to symmetric surfaces for CCNC assembly, deviating from what is observed in prior structures using artificial sequences. At mitosis, we propose that CCNC asymmetry accommodates its asymmetric connections at the chromosome/kinetochore interface. VIDEO ABSTRACT.
History
DepositionOct 23, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 24, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 31, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_abbrev / _citation.journal_volume ..._citation.journal_abbrev / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.title
Revision 1.2Aug 14, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Sep 4, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 2.0Oct 2, 2019Group: Data collection / Polymer sequence / Category: entity_poly / Item: _entity_poly.type
Revision 2.1Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
A: Histone H3-like centromeric protein A
B: Histone H4
C: Histone H2A type 1-C
D: Histone H2B type 2-F
E: Histone H3-like centromeric protein A
F: Histone H4
G: Histone H2A type 1-C
H: Histone H2B type 2-F
I: DNA/RNA (147-MER)
J: DNA/RNA (147-MER)
K: Centromere protein C
L: Centromere protein C
M: Centromere protein N


Theoretical massNumber of molelcules
Total (without water)209,47813
Polymers209,47813
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: cross-linking
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area59880 Å2
ΔGint-393 kcal/mol
Surface area89720 Å2

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Components

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Protein , 4 types, 8 molecules AEBFCGDH

#1: Protein Histone H3-like centromeric protein A / Centromere autoantigen A / Centromere protein A / CENP-A


Mass: 11993.037 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CENPA
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P49450
#2: Protein Histone H4 /


Mass: 10604.521 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: HIST1H4A, H4/A, H4FA, HIST1H4B, H4/I, H4FI, HIST1H4C, H4/G, H4FG, HIST1H4D, H4/B, H4FB, HIST1H4E, H4/J, H4FJ, HIST1H4F, H4/C, H4FC, HIST1H4H, H4/H, H4FH, HIST1H4I, H4/M, H4FM, HIST1H4J, H4/E, ...Gene: HIST1H4A, H4/A, H4FA, HIST1H4B, H4/I, H4FI, HIST1H4C, H4/G, H4FG, HIST1H4D, H4/B, H4FB, HIST1H4E, H4/J, H4FJ, HIST1H4F, H4/C, H4FC, HIST1H4H, H4/H, H4FH, HIST1H4I, H4/M, H4FM, HIST1H4J, H4/E, H4FE, HIST1H4K, H4/D, H4FD, HIST1H4L, H4/K, H4FK, HIST2H4A, H4/N, H4F2, H4FN, HIST2H4, HIST2H4B, H4/O, H4FO, HIST4H4
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P62805
#3: Protein Histone H2A type 1-C / Histone H2A/l


Mass: 11494.393 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HIST1H2AC, H2AFL
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q93077
#4: Protein Histone H2B type 2-F


Mass: 10249.723 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HIST2H2BF
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q5QNW6

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DNA/RNA (147- ... , 2 types, 2 molecules IJ

#5: DNA chain DNA/RNA (147-MER)


Mass: 45141.918 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#6: DNA chain DNA/RNA (147-MER)


Mass: 45582.188 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Centromere protein ... , 2 types, 3 molecules KLM

#7: Protein/peptide 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: 2508.834 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: Q03188
#8: Protein Centromere protein N / / CENP-N / Interphase centromere complex protein 32


Mass: 25052.885 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: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q96H22

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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: CENP-A chromatin complex bound with CENP-C and CENP-N of CCAN kinetochore components
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1-#8 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.276 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 7.5 / Details: 10 mM HEPES, 50 mN NaCl, 1 mM EDTA, 1mM DTT
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3 4C
VitrificationInstrument: LEICA EM CPC / Cryogen name: ETHANE / Details: Blot for 8 seconds before plunging

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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: OTHER / Nominal magnification: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 2
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
2SerialEM3image acquisition
4Gctf1.06CTF correction
7PHENIX1.13model fitting
8Coot1.13model fitting
10RELION2.1initial Euler assignment
11RELION3final Euler assignment
12RELION2.1classification
13RELION33D reconstruction
14PHENIX1.13model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 113950 / Symmetry type: POINT

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