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- PDB-7u4d: CryoEM structure of CENP-N promoted nucleosome stacks with CENP-A... -

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Basic information

Entry
Database: PDB / ID: 7u4d
TitleCryoEM structure of CENP-N promoted nucleosome stacks with CENP-A and 601 DNA sequence
Components
  • (DNA (147-MER)) x 2
  • Centromere protein N
  • Histone H2A
  • Histone H2B type 1-C/E/F/G/I
  • Histone H3-like centromeric protein A
  • Histone H4
KeywordsDNA BINDING PROTEIN/DNA / nucleosome / CENP-A / kinetochore / CENP-N / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


inner kinetochore / protein localization to chromosome, centromeric region / CENP-A containing chromatin assembly / condensed chromosome, centromeric region / kinetochore assembly / mitotic cytokinesis / chromosome, centromeric region / mitotic metaphase chromosome alignment / pericentric heterochromatin / negative regulation of megakaryocyte differentiation ...inner kinetochore / protein localization to chromosome, centromeric region / CENP-A containing chromatin assembly / condensed chromosome, centromeric region / kinetochore assembly / mitotic cytokinesis / chromosome, centromeric region / mitotic metaphase chromosome alignment / pericentric heterochromatin / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / Replacement of protamines by nucleosomes in the male pronucleus / CENP-A containing nucleosome / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Packaging Of Telomere Ends / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / telomere organization / ChAHP complex assembly / Deposition of new CENPA-containing nucleosomes at the centromere / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / RNA Polymerase I Promoter Opening / Inhibition of DNA recombination at telomere / Resolution of Sister Chromatid Cohesion / Assembly of the ORC complex at the origin of replication / FXIIa activates plasma kallikrein-kinin system / chromosome segregation / SUMOylation of chromatin organization proteins / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / Meiotic synapsis / DNA methylation / Condensation of Prophase Chromosomes / Chromatin modifications during the maternal to zygotic transition (MZT) / HCMV Late Events / SIRT1 negatively regulates rRNA expression / NuRD complex assembly / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / Interaction of NuRD complexes with transcription factors / PRC2 methylates histones and DNA / Regulation of endogenous retroelements by KRAB-ZFP proteins / Defective pyroptosis / CHD1 and CHD2 subfamily / establishment of mitotic spindle orientation / HDACs deacetylate histones / CHD6, CHD7, CHD8, CHD9 subfamily / Transcriptional regulation by small RNAs / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / RNA Polymerase I Promoter Escape / Nonhomologous End-Joining (NHEJ) / RHO GTPases Activate Formins / HDMs demethylate histones / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / Negative Regulation of CDH1 Gene Transcription / NoRC negatively regulates rRNA expression / PKMTs methylate histone lysines / G2/M DNA damage checkpoint / Formation of the beta-catenin:TCF transactivating complex / B-WICH complex positively regulates rRNA expression / DNA Damage/Telomere Stress Induced Senescence / Meiotic recombination / kinetochore / Pre-NOTCH Transcription and Translation / Activation of anterior HOX genes in hindbrain development during early embryogenesis / Transcriptional regulation of granulopoiesis / nucleosomal DNA binding / RMTs methylate histone arginines / HCMV Early Events / Metalloprotease DUBs / innate immune response in mucosa / structural constituent of chromatin / Separation of Sister Chromatids / Regulation of PD-L1(CD274) transcription / nucleosome / nucleosome assembly / UCH proteinases / HATs acetylate histones / E3 ubiquitin ligases ubiquitinate target proteins / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / MLL4 and MLL3 complexes regulate expression of PPARG target genes in adipogenesis and hepatic steatosis / chromatin organization / RUNX1 regulates transcription of genes involved in differentiation of HSCs / Processing of DNA double-strand break ends / Dengue Virus-Host Interactions / antimicrobial humoral immune response mediated by antimicrobial peptide / Senescence-Associated Secretory Phenotype (SASP) / heterochromatin formation / Oxidative Stress Induced Senescence / antibacterial humoral response / Estrogen-dependent gene expression / chromosome, telomeric region / defense response to Gram-positive bacterium / Ub-specific processing proteases / negative regulation of cell population proliferation / protein heterodimerization activity / Amyloid fiber formation / chromatin binding
Similarity search - Function
: / Centromere protein Chl4/mis15/CENP-N / Centromere protein Chl4/mis15/CENP-N / : / Histone H2A conserved site / Histone H2A signature. / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A, C-terminal domain ...: / Centromere protein Chl4/mis15/CENP-N / Centromere protein Chl4/mis15/CENP-N / : / Histone H2A conserved site / Histone H2A signature. / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone 2A / Histone H2A / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 domain / Histone-fold
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: 8.1 Å
AuthorsZhou, K. / Luger, K.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: CENP-N promotes the compaction of centromeric chromatin.
Authors: Keda Zhou / Magdalena Gebala / Dustin Woods / Kousik Sundararajan / Garrett Edwards / Dan Krzizike / Jeff Wereszczynski / Aaron F Straight / Karolin Luger /
Abstract: The histone variant CENP-A is the epigenetic determinant for the centromere, where it is interspersed with canonical H3 to form a specialized chromatin structure that nucleates the kinetochore. How ...The histone variant CENP-A is the epigenetic determinant for the centromere, where it is interspersed with canonical H3 to form a specialized chromatin structure that nucleates the kinetochore. How nucleosomes at the centromere arrange into higher order structures is unknown. Here we demonstrate that the human CENP-A-interacting protein CENP-N promotes the stacking of CENP-A-containing mononucleosomes and nucleosomal arrays through a previously undefined interaction between the α6 helix of CENP-N with the DNA of a neighboring nucleosome. We describe the cryo-EM structures and biophysical characterization of such CENP-N-mediated nucleosome stacks and nucleosomal arrays and demonstrate that this interaction is responsible for the formation of densely packed chromatin at the centromere in the cell. Our results provide first evidence that CENP-A, together with CENP-N, promotes specific chromatin higher order structure at the centromere.
History
DepositionFeb 28, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 30, 2022Provider: repository / Type: Initial release
Revision 1.1Apr 20, 2022Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.year
Revision 1.2May 4, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.3Feb 21, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
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Assembly

Deposited unit
A: Histone H3-like centromeric protein A
B: Histone H4
C: Histone H2A
D: Histone H2B type 1-C/E/F/G/I
E: Histone H3-like centromeric protein A
F: Histone H4
G: Histone H2A
H: Histone H2B type 1-C/E/F/G/I
I: DNA (147-MER)
J: DNA (147-MER)
K: Centromere protein N
L: Histone H3-like centromeric protein A
M: Histone H4
N: Histone H2A
O: Histone H2B type 1-C/E/F/G/I
P: Histone H3-like centromeric protein A
Q: Histone H4
R: Histone H2A
S: Histone H2B type 1-C/E/F/G/I
T: DNA (147-MER)
U: DNA (147-MER)
V: Centromere protein N


Theoretical massNumber of molelcules
Total (without water)473,20022
Polymers473,20022
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization, Analytical Ultracentrifugation
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 5 types, 18 molecules AELPBFMQCGNRDHOSKV

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


Mass: 16023.630 Da / Num. of mol.: 4
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: 11394.426 Da / Num. of mol.: 4
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.: 4
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 type 1-C/E/F/G/I / 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.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: H2BC4, H2BFL, HIST1H2BC, H2BC6, H2BFH, HIST1H2BE, H2BC7, H2BFG, HIST1H2BF, H2BC8, H2BFA, HIST1H2BG, H2BC10, H2BFK, HIST1H2BI
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.: 2
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, 4 molecules ITJU

#5: DNA chain DNA (147-MER)


Mass: 45153.781 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#6: DNA chain DNA (147-MER)


Mass: 45594.043 Da / Num. of mol.: 2 / 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: 3D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: HUMAN KINETOCHORE PROTEIN CENP-N WITH THE CENTROMERIC NUCLEOSOME CONTAINING CENP-A (601 sequence DNA)
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 8.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 174936 / Symmetry type: POINT
RefinementHighest resolution: 8.1 Å

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