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- PDB-8kb5: Cryo-EM structure of the human nucleosome containing H3.8 -

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Entry
Database: PDB / ID: 8kb5
TitleCryo-EM structure of the human nucleosome containing H3.8
Components
  • (DNA (145-MER)) x 2
  • Histone H2A type 1-B/E
  • Histone H2B type 1-J
  • Histone H3.8
  • Histone H4
KeywordsDNA BINDING PROTEIN / complex / chromatin / nucleosome / gene regulation
Function / homology
Function and homology information


negative regulation of tumor necrosis factor-mediated signaling pathway / 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 / Packaging Of Telomere Ends / 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 / Recognition and association of DNA glycosylase with site containing an affected pyrimidine ...negative regulation of tumor necrosis factor-mediated signaling pathway / 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 / Packaging Of Telomere Ends / 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 / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / telomere organization / Inhibition of DNA recombination at telomere / RNA Polymerase I Promoter Opening / Meiotic synapsis / Assembly of the ORC complex at the origin of replication / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / SUMOylation of chromatin organization proteins / DNA methylation / Condensation of Prophase Chromosomes / Chromatin modifications during the maternal to zygotic transition (MZT) / SIRT1 negatively regulates rRNA expression / HCMV Late Events / innate immune response in mucosa / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / PRC2 methylates histones and DNA / Regulation of endogenous retroelements by KRAB-ZFP proteins / Defective pyroptosis / HDACs deacetylate histones / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / Nonhomologous End-Joining (NHEJ) / lipopolysaccharide binding / RNA Polymerase I Promoter Escape / Transcriptional regulation by small RNAs / Formation of the beta-catenin:TCF transactivating complex / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / HDMs demethylate histones / G2/M DNA damage checkpoint / NoRC negatively regulates rRNA expression / DNA Damage/Telomere Stress Induced Senescence / B-WICH complex positively regulates rRNA expression / PKMTs methylate histone lysines / Meiotic recombination / Pre-NOTCH Transcription and Translation / Metalloprotease DUBs / RMTs methylate histone arginines / Activation of anterior HOX genes in hindbrain development during early embryogenesis / Transcriptional regulation of granulopoiesis / HCMV Early Events / antimicrobial humoral immune response mediated by antimicrobial peptide / structural constituent of chromatin / UCH proteinases / antibacterial humoral response / nucleosome / heterochromatin formation / nucleosome assembly / 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 / HATs acetylate histones / chromatin organization / MLL4 and MLL3 complexes regulate expression of PPARG target genes in adipogenesis and hepatic steatosis / Processing of DNA double-strand break ends / Senescence-Associated Secretory Phenotype (SASP) / Oxidative Stress Induced Senescence / defense response to Gram-negative bacterium / killing of cells of another organism / Estrogen-dependent gene expression / chromosome, telomeric region / defense response to Gram-positive bacterium / Ub-specific processing proteases / Amyloid fiber formation / protein heterodimerization activity / negative regulation of cell population proliferation / protein-containing complex / extracellular space / DNA binding / RNA binding / extracellular exosome / extracellular region / nucleoplasm / nucleus / membrane / cytosol
Similarity search - Function
: / Histone H2B signature. / Histone H2A conserved site / Histone H2A signature. / Histone H2B / Histone H2B / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone 2A / Histone H2A ...: / Histone H2B signature. / Histone H2A conserved site / Histone H2A 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 H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 domain / Histone-fold
Similarity search - Domain/homology
: / DNA / DNA (> 10) / DNA (> 100) / Histone H2A type 1-B/E / Histone H2B type 1-J / Histone H4
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.26133 Å
AuthorsHirai, H. / Kujirai, T. / Takizawa, Y. / Kurumizaka, H.
Funding support Japan, 8items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP22K06098 Japan
Japan Society for the Promotion of Science (JSPS)JP22K15033 Japan
Japan Society for the Promotion of Science (JSPS)JP20H03201 Japan
Japan Society for the Promotion of Science (JSPS)JP20H05690 Japan
Japan Society for the Promotion of Science (JSPS)JP22KJ0858 Japan
Japan Society for the Promotion of Science (JSPS)JP23H05475 Japan
Japan Science and TechnologyJPMJER1901 Japan
Japan Agency for Medical Research and Development (AMED)JP23ama121009 Japan
CitationJournal: J Biochem / Year: 2023
Title: Cryo-EM and biochemical analyses of the nucleosome containing the human histone H3 variant H3.8.
Authors: Seiya Hirai / Tomoya Kujirai / Munetaka Akatsu / Mitsuo Ogasawara / Haruhiko Ehara / Shun-Ichi Sekine / Yasuyuki Ohkawa / Yoshimasa Takizawa / Hitoshi Kurumizaka /
Abstract: Histone H3.8 is a non-allelic human histone H3 variant derived from H3.3. H3.8 reportedly forms an unstable nucleosome, but its structure and biochemical characteristics have not been revealed yet. ...Histone H3.8 is a non-allelic human histone H3 variant derived from H3.3. H3.8 reportedly forms an unstable nucleosome, but its structure and biochemical characteristics have not been revealed yet. In the present study, we reconstituted the nucleosome containing H3.8. Consistent with previous results, the H3.8 nucleosome is thermally unstable as compared to the H3.3 nucleosome. The entry/exit DNA regions of the H3.8 nucleosome are more accessible to micrococcal nuclease than those of the H3.3 nucleosome. Nucleosome transcription assays revealed that the RNA polymerase II (RNAPII) pausing around the superhelical location (SHL) -1 position, which is about 60 base pairs from the nucleosomal DNA entry site, is drastically alleviated. On the other hand, the RNAPII pausing around the SHL(-5) position, which is about 20 base pairs from the nucleosomal DNA entry site, is substantially increased. The cryo-electron microscopy structure of the H3.8 nucleosome explains the mechanisms of the enhanced accessibility of the entry/exit DNA regions, reduced thermal stability and altered RNAPII transcription profile.
History
DepositionAug 3, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 18, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 27, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Histone H3.8
B: Histone H4
C: Histone H2A type 1-B/E
D: Histone H2B type 1-J
E: Histone H3.8
F: Histone H4
G: Histone H2A type 1-B/E
H: Histone H2B type 1-J
I: DNA (145-MER)
J: DNA (145-MER)


Theoretical massNumber of molelcules
Total (without water)203,18710
Polymers203,18710
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: native gel electrophoresis, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 4 types, 8 molecules AEBFCGDH

#1: Protein Histone H3.8


Mass: 16494.197 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: H3F3AP5 / Production host: Escherichia coli (E. coli)
#2: Protein Histone H4


Mass: 11676.703 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: H4C1 / Production host: Escherichia coli (E. coli) / References: UniProt: P62805
#3: Protein Histone H2A type 1-B/E / Histone H2A.2 / Histone H2A/a / Histone H2A/m


Mass: 14447.825 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: H2AC4, H2AFM, HIST1H2AB, H2AC8, H2AFA, HIST1H2AE / Production host: Escherichia coli (E. coli) / References: UniProt: P04908
#4: Protein Histone H2B type 1-J / Histone H2B.1 / Histone H2B.r / H2B/r


Mass: 14217.516 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: H2BC11, H2BFR, HIST1H2BJ / Production host: Escherichia coli (E. coli) / References: UniProt: P06899

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain DNA (145-MER)


Mass: 44761.523 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli) / References: GenBank: 2041866466
#6: DNA chain DNA (145-MER)


Mass: 44752.508 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli) / References: GenBank: 2041866466

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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: The human nucleosome containing H3.8 / Type: COMPLEX
Details: Human nucleosome with 145 bp Widom 601L DNA and H3.8
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Human nucleosome with 145 bp Widom601L DNA and H3.8
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277 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 FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 61.2 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
10RELION4initial Euler assignment
11RELION4final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.26133 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 3900336 / Symmetry type: POINT

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