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- EMDB-37070: Cryo-EM structure of the human nucleosome containing H3.8 -

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Entry
Database: EMDB / ID: EMD-37070
TitleCryo-EM structure of the human nucleosome containing H3.8
Map data
Sample
  • Complex: The human nucleosome containing H3.8
    • Protein or peptide: Histone H3.8
    • Protein or peptide: Histone H4
    • Protein or peptide: Histone H2A type 1-B/E
    • Protein or peptide: Histone H2B type 1-J
    • DNA: DNA (145-MER)
    • DNA: DNA (145-MER)
Keywordscomplex / chromatin / nucleosome / gene regulation / DNA BINDING PROTEIN
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 / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged 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 / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Deposition of new CENPA-containing nucleosomes at the centromere / telomere organization / Inhibition of DNA recombination at telomere / Meiotic synapsis / RNA Polymerase I Promoter Opening / Assembly of the ORC complex at the origin of replication / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / innate immune response in mucosa / SUMOylation of chromatin organization proteins / DNA methylation / Condensation of Prophase Chromosomes / Chromatin modifications during the maternal to zygotic transition (MZT) / HCMV Late Events / SIRT1 negatively regulates rRNA expression / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / PRC2 methylates histones and DNA / Regulation of endogenous retroelements by KRAB-ZFP proteins / Defective pyroptosis / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / HDACs deacetylate histones / Nonhomologous End-Joining (NHEJ) / RNA Polymerase I Promoter Escape / lipopolysaccharide binding / 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 / G2/M DNA damage checkpoint / HDMs demethylate histones / 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 / antibacterial humoral response / UCH proteinases / nucleosome / heterochromatin formation / E3 ubiquitin ligases ubiquitinate target proteins / nucleosome assembly / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / chromatin organization / HATs acetylate histones / RUNX1 regulates transcription of genes involved in differentiation of HSCs / 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) / defense response to Gram-negative bacterium / Oxidative Stress Induced Senescence / 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 / DNA binding / extracellular space / RNA binding / extracellular exosome / extracellular region / nucleoplasm / nucleus / membrane / cytosol
Similarity search - Function
: / 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 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 / 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 / Histone-fold
Similarity search - Domain/homology
Histone H2A type 1-B/E / Histone H2B type 1-J / Histone H4
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.26133 Å
AuthorsHirai H / Kujirai T / Takizawa Y / Kurumizaka H
Funding support Japan, 8 items
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, 2023-
Header (metadata) releaseOct 18, 2023-
Map releaseOct 18, 2023-
UpdateDec 27, 2023-
Current statusDec 27, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_37070.png

Downloads & links

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Map

FileDownload / File: emd_37070.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 256 pix.
= 271.36 Å		1.06 Å/pix.
x 256 pix.
= 271.36 Å		1.06 Å/pix.
x 256 pix.
= 271.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00559
Minimum - Maximum-0.078746885 - 0.19291991
Average (Standard dev.)0.00012428322 (±0.002883337)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 271.36 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_37070_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_37070_half_map_2.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : The human nucleosome containing H3.8

EntireName: The human nucleosome containing H3.8
Components
  • Complex: The human nucleosome containing H3.8
    • Protein or peptide: Histone H3.8
    • Protein or peptide: Histone H4
    • Protein or peptide: Histone H2A type 1-B/E
    • Protein or peptide: Histone H2B type 1-J
    • DNA: DNA (145-MER)
    • DNA: DNA (145-MER)

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Supramolecule #1: The human nucleosome containing H3.8

SupramoleculeName: The human nucleosome containing H3.8 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Human nucleosome with 145 bp Widom 601L DNA and H3.8
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Histone H3.8

MacromoleculeName: Histone H3.8 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 16.494197 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSHMARTRWT ARKSTGGIAP RKQLATKATC KSAPSTGGVK KPHRYRPGTV ALREIRHYQK STELLIRKLP FQRLVREIAQ DFKTDLRFQ RAAIGTLQEA SEAYLVGLFE DTNLCAIHAT RVTIMPKDIQ LARHIRGERV SEYTMM

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Macromolecule #2: Histone H4

MacromoleculeName: Histone H4 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.676703 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSHMSGRGKG GKGLGKGGAK RHRKVLRDNI QGITKPAIRR LARRGGVKRI SGLIYEETRG VLKVFLENVI RDAVTYTEHA KRKTVTAMD VVYALKRQGR TLYGFGG

UniProtKB: Histone H4

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Macromolecule #3: Histone H2A type 1-B/E

MacromoleculeName: Histone H2A type 1-B/E / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 14.447825 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSHMSGRGKQ GGKARAKAKT RSSRAGLQFP VGRVHRLLRK GNYSERVGAG APVYLAAVLE YLTAEILELA GNAARDNKKT RIIPRHLQL AIRNDEELNK LLGRVTIAQG GVLPNIQAVL LPKKTESHHK AKGK

UniProtKB: Histone H2A type 1-B/E

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Macromolecule #4: Histone H2B type 1-J

MacromoleculeName: Histone H2B type 1-J / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 14.217516 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSHMPEPAKS APAPKKGSKK AVTKAQKKDG KKRKRSRKES YSIYVYKVLK QVHPDTGISS KAMGIMNSFV NDIFERIAGE ASRLAHYNK RSTITSREIQ TAVRLLLPGE LAKHAVSEGT KAVTKYTSAK

UniProtKB: Histone H2B type 1-J

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Macromolecule #5: DNA (145-MER)

MacromoleculeName: DNA (145-MER) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 44.761523 KDa
SequenceString: (DA)(DT)(DC)(DA)(DC)(DA)(DA)(DT)(DC)(DC) (DC)(DG)(DG)(DT)(DG)(DC)(DC)(DG)(DA)(DG) (DG)(DC)(DC)(DG)(DC)(DT)(DC)(DA)(DA) (DT)(DT)(DG)(DG)(DT)(DC)(DG)(DT)(DA)(DG) (DA) (DC)(DA)(DG)(DC)(DT)(DC) ...String:
(DA)(DT)(DC)(DA)(DC)(DA)(DA)(DT)(DC)(DC) (DC)(DG)(DG)(DT)(DG)(DC)(DC)(DG)(DA)(DG) (DG)(DC)(DC)(DG)(DC)(DT)(DC)(DA)(DA) (DT)(DT)(DG)(DG)(DT)(DC)(DG)(DT)(DA)(DG) (DA) (DC)(DA)(DG)(DC)(DT)(DC)(DT)(DA) (DG)(DC)(DA)(DC)(DC)(DG)(DC)(DT)(DT)(DA) (DA)(DA) (DC)(DG)(DC)(DA)(DC)(DG)(DT) (DA)(DC)(DG)(DG)(DA)(DA)(DT)(DC)(DC)(DG) (DT)(DA)(DC) (DG)(DT)(DG)(DC)(DG)(DT) (DT)(DT)(DA)(DA)(DG)(DC)(DG)(DG)(DT)(DG) (DC)(DT)(DA)(DG) (DA)(DG)(DC)(DT)(DG) (DT)(DC)(DT)(DA)(DC)(DG)(DA)(DC)(DC)(DA) (DA)(DT)(DT)(DG)(DA) (DG)(DC)(DG)(DG) (DC)(DC)(DT)(DC)(DG)(DG)(DC)(DA)(DC)(DC) (DG)(DG)(DG)(DA)(DT)(DT) (DG)(DT)(DG) (DA)(DT)

GENBANK: GENBANK: OU026066.1

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Macromolecule #6: DNA (145-MER)

MacromoleculeName: DNA (145-MER) / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 44.752508 KDa
SequenceString: (DA)(DT)(DC)(DA)(DC)(DA)(DA)(DT)(DC)(DC) (DC)(DG)(DG)(DT)(DG)(DC)(DC)(DG)(DA)(DG) (DG)(DC)(DC)(DG)(DC)(DT)(DC)(DA)(DA) (DT)(DT)(DG)(DG)(DT)(DC)(DG)(DT)(DA)(DG) (DA) (DC)(DA)(DG)(DC)(DT)(DC) ...String:
(DA)(DT)(DC)(DA)(DC)(DA)(DA)(DT)(DC)(DC) (DC)(DG)(DG)(DT)(DG)(DC)(DC)(DG)(DA)(DG) (DG)(DC)(DC)(DG)(DC)(DT)(DC)(DA)(DA) (DT)(DT)(DG)(DG)(DT)(DC)(DG)(DT)(DA)(DG) (DA) (DC)(DA)(DG)(DC)(DT)(DC)(DT)(DA) (DG)(DC)(DA)(DC)(DC)(DG)(DC)(DT)(DT)(DA) (DA)(DA) (DC)(DG)(DC)(DA)(DC)(DG)(DT) (DA)(DC)(DG)(DG)(DA)(DT)(DT)(DC)(DC)(DG) (DT)(DA)(DC) (DG)(DT)(DG)(DC)(DG)(DT) (DT)(DT)(DA)(DA)(DG)(DC)(DG)(DG)(DT)(DG) (DC)(DT)(DA)(DG) (DA)(DG)(DC)(DT)(DG) (DT)(DC)(DT)(DA)(DC)(DG)(DA)(DC)(DC)(DA) (DA)(DT)(DT)(DG)(DA) (DG)(DC)(DG)(DG) (DC)(DC)(DT)(DC)(DG)(DG)(DC)(DA)(DC)(DC) (DG)(DG)(DG)(DA)(DT)(DT) (DG)(DT)(DG) (DA)(DT)

GENBANK: GENBANK: OU026066.1

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 20 sec.
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsHuman nucleosome with 145 bp Widom601L DNA and H3.8

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 61.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3ut9

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.26133 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 3900336
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
FSC plot (resolution estimation)

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