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- PDB-6c0w: Cryo-EM structure of human kinetochore protein CENP-N with the ce... -

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Entry
Database: PDB / ID: 6c0w
TitleCryo-EM structure of human kinetochore protein CENP-N with the centromeric nucleosome containing CENP-A
Components
  • (147 mer DNA) x 2
  • Centromere protein N
  • Histone H2A
  • Histone H2B
  • Histone H3-like centromeric protein A
  • Histone H4
KeywordsSTRUCTURAL PROTEIN/DNA / Nucleosome / CENP-A / kinetochore / CENP-N / STRUCTURAL PROTEIN-DNA complex
Function / homology
Function and homology information


inner kinetochore / CENP-A containing chromatin assembly / protein localization to chromosome, centromeric region / kinetochore assembly / condensed chromosome, centromeric region / establishment of mitotic spindle orientation / mitotic cytokinesis / chromosome, centromeric region / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin ...inner kinetochore / CENP-A containing chromatin assembly / protein localization to chromosome, centromeric region / kinetochore assembly / condensed chromosome, centromeric region / establishment of mitotic spindle orientation / mitotic cytokinesis / chromosome, centromeric region / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / pericentric heterochromatin / Replacement of protamines by nucleosomes in the male pronucleus / CENP-A containing nucleosome / Packaging Of Telomere Ends / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / Deposition of new CENPA-containing nucleosomes at the centromere / Resolution of Sister Chromatid Cohesion / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Inhibition of DNA recombination at telomere / Meiotic synapsis / telomere organization / RNA Polymerase I Promoter Opening / Assembly of the ORC complex at the origin of replication / SUMOylation of chromatin organization proteins / DNA methylation / Condensation of Prophase Chromosomes / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / Chromatin modifications during the maternal to zygotic transition (MZT) / SIRT1 negatively regulates rRNA expression / HCMV Late Events / innate immune response in mucosa / PRC2 methylates histones and DNA / Defective pyroptosis / chromosome segregation / RHO GTPases Activate Formins / HDACs deacetylate histones / RNA Polymerase I Promoter Escape / Nonhomologous End-Joining (NHEJ) / Transcriptional regulation by small RNAs / Formation of the beta-catenin:TCF transactivating complex / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / NoRC negatively regulates rRNA expression / G2/M DNA damage checkpoint / HDMs demethylate histones / B-WICH complex positively regulates rRNA expression / DNA Damage/Telomere Stress Induced Senescence / Metalloprotease DUBs / PKMTs methylate histone lysines / RMTs methylate histone arginines / Meiotic recombination / Pre-NOTCH Transcription and Translation / nucleosome assembly / Activation of anterior HOX genes in hindbrain development during early embryogenesis / HCMV Early Events / Transcriptional regulation of granulopoiesis / structural constituent of chromatin / Separation of Sister Chromatids / UCH proteinases / nucleosome / antimicrobial humoral immune response mediated by antimicrobial peptide / E3 ubiquitin ligases ubiquitinate target proteins / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / RUNX1 regulates transcription of genes involved in differentiation of HSCs / chromatin organization / Processing of DNA double-strand break ends / HATs acetylate histones / antibacterial humoral response / Senescence-Associated Secretory Phenotype (SASP) / Oxidative Stress Induced Senescence / Estrogen-dependent gene expression / chromosome, telomeric region / Ub-specific processing proteases / defense response to Gram-positive bacterium / protein heterodimerization activity / Amyloid fiber formation / negative regulation of cell population proliferation / chromatin binding / protein-containing complex / DNA binding / extracellular space / RNA binding / extracellular exosome / extracellular region / nucleoplasm / membrane / identical protein binding / nucleus / cytosol
Similarity search - Function
Centromere protein Chl4/mis15/CENP-N / Kinetochore protein CHL4 like / Histone, subunit A / Histone, subunit A / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / Histone H2A, C-terminal domain ...Centromere protein Chl4/mis15/CENP-N / Kinetochore protein CHL4 like / Histone, subunit A / Histone, subunit A / 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 H2A / Histone 2A / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Histone-fold / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / Histone H3-like centromeric protein A / Histone H4 / Histone H2B type 1-C/E/F/G/I / Histone H2A type 1-C / Centromere protein N
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsZhou, K. / Pentakota, S. / Vetter, I.R. / Morgan, G.P. / Petrovic, A. / Musacchio, A. / Luger, K.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM067777 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Elife / Year: 2017
Title: Decoding the centromeric nucleosome through CENP-N.
Authors: Satyakrishna Pentakota / Keda Zhou / Charlotte Smith / Stefano Maffini / Arsen Petrovic / Garry P Morgan / John R Weir / Ingrid R Vetter / Andrea Musacchio / Karolin Luger /
Abstract: Centromere protein (CENP) A, a histone H3 variant, is a key epigenetic determinant of chromosome domains known as centromeres. Centromeres nucleate kinetochores, multi-subunit complexes that capture ...Centromere protein (CENP) A, a histone H3 variant, is a key epigenetic determinant of chromosome domains known as centromeres. Centromeres nucleate kinetochores, multi-subunit complexes that capture spindle microtubules to promote chromosome segregation during mitosis. Two kinetochore proteins, CENP-C and CENP-N, recognize CENP-A in the context of a rare CENP-A nucleosome. Here, we reveal the structural basis for the exquisite selectivity of CENP-N for centromeres. CENP-N uses charge and space complementarity to decode the L1 loop that is unique to CENP-A. It also engages in extensive interactions with a 15-base pair segment of the distorted nucleosomal DNA double helix, in a position predicted to exclude chromatin remodelling enzymes. Besides CENP-A, stable centromere recruitment of CENP-N requires a coincident interaction with a newly identified binding motif on nucleosome-bound CENP-C. Collectively, our studies clarify how CENP-N and CENP-C decode and stabilize the non-canonical CENP-A nucleosome to enforce epigenetic centromere specification and kinetochore assembly.
History
DepositionJan 2, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 17, 2018Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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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
D: Histone H2B
E: Histone H3-like centromeric protein A
F: Histone H4
G: Histone H2A
H: Histone H2B
I: 147 mer DNA
J: 147 mer DNA
K: Centromere protein N


Theoretical massNumber of molelcules
Total (without water)236,48611
Polymers236,48611
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area52740 Å2
ΔGint-388 kcal/mol
Surface area83700 Å2

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Components

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Protein , 5 types, 9 molecules AEBFCGDHK

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


Mass: 16023.630 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: 11337.374 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 / / Histone H2A/l


Mass: 14135.523 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 (E. coli) / References: UniProt: Q93077
#4: Protein Histone H2B / / Histone H2B.1 A / Histone H2B.a / H2B/a / Histone H2B.g / H2B/g / Histone H2B.h / H2B/h / Histone ...Histone H2B.1 A / Histone H2B.a / H2B/a / Histone H2B.g / H2B/g / Histone H2B.h / H2B/h / Histone H2B.k / H2B/k / Histone H2B.l / H2B/l


Mass: 13937.213 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: HIST1H2BC, H2BFL, HIST1H2BE, H2BFH, HIST1H2BF, H2BFG, HIST1H2BG, H2BFA, HIST1H2BI, H2BFK
Production host: Escherichia coli (E. coli) / References: UniProt: P62807
#7: Protein Centromere protein N / / CENP-N / Interphase centromere complex protein 32


Mass: 34870.695 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: Insect cell expression vector pTIE1 (others)
References: UniProt: Q96H22

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain 147 mer DNA


Mass: 45153.781 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#6: DNA chain 147 mer DNA


Mass: 45594.043 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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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: complex of CENP-A nucleosome with CENP-N / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.23 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-Cl1
225 mMsodium chlorideNaClSodium chloride1
31 mMDTT1
41 mMEDTAEthylenediaminetetraacetic acid1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277.15 K

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 29000 X / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 1.3 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
4GctfCTF correction
7UCSF Chimeramodel fitting
8Cootmodel fitting
10PHENIXmodel refinement
13RELION2.1b1classification
14RELION2.1b13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1843269
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 937118 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00613936
ELECTRON MICROSCOPYf_angle_d0.79320016
ELECTRON MICROSCOPYf_dihedral_angle_d23.8247376
ELECTRON MICROSCOPYf_chiral_restr0.0472270
ELECTRON MICROSCOPYf_plane_restr0.0061559

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