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- EMDB-44148: The cryo-EM structure of the H2A.Z-H3.3 double-variant nucleosome -

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Basic information

Entry
Database: EMDB / ID: EMD-44148
TitleThe cryo-EM structure of the H2A.Z-H3.3 double-variant nucleosome
Map data
Sample
  • Complex: nucleosome containing histone variants H3.3 and H2A.Z
    • Protein or peptide: Histone H3.3
    • Protein or peptide: Histone H4
    • Protein or peptide: Histone H2A.Z
    • Protein or peptide: Histone H2B 1.1
    • DNA: DNA (128-MER)
    • DNA: DNA (128-MER)
Keywordshistone variant / nucleosome / chromatin / complex / STRUCTURAL PROTEIN / STRUCTURAL PROTEIN-DNA complex
Function / homology
Function and homology information


negative regulation of chromosome condensation / Barr body / regulation of centromere complex assembly / pericentric heterochromatin formation / inner kinetochore / muscle cell differentiation / oocyte maturation / nucleosomal DNA binding / nucleus organization / spermatid development ...negative regulation of chromosome condensation / Barr body / regulation of centromere complex assembly / pericentric heterochromatin formation / inner kinetochore / muscle cell differentiation / oocyte maturation / nucleosomal DNA binding / nucleus organization / spermatid development / single fertilization / subtelomeric heterochromatin formation / heterochromatin / RNA polymerase II core promoter sequence-specific DNA binding / Replacement of protamines by nucleosomes in the male pronucleus / telomere organization / embryo implantation / Inhibition of DNA recombination at telomere / 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 / DNA methylation / Condensation of Prophase Chromosomes / Chromatin modifications during the maternal to zygotic transition (MZT) / 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) / RNA Polymerase I Promoter Escape / cellular response to estradiol stimulus / Transcriptional regulation by small RNAs / Formation of the beta-catenin:TCF transactivating complex / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / euchromatin / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / NoRC negatively regulates rRNA expression / multicellular organism growth / B-WICH complex positively regulates rRNA expression / Meiotic recombination / Pre-NOTCH Transcription and Translation / Activation of anterior HOX genes in hindbrain development during early embryogenesis / Transcriptional regulation of granulopoiesis / nucleosome assembly / male gonad development / osteoblast differentiation / structural constituent of chromatin / nucleosome / chromatin organization / RUNX1 regulates transcription of genes involved in differentiation of HSCs / Factors involved in megakaryocyte development and platelet production / MLL4 and MLL3 complexes regulate expression of PPARG target genes in adipogenesis and hepatic steatosis / positive regulation of cell growth / Senescence-Associated Secretory Phenotype (SASP) / Oxidative Stress Induced Senescence / Estrogen-dependent gene expression / chromosome, telomeric region / cell population proliferation / RNA polymerase II cis-regulatory region sequence-specific DNA binding / protein heterodimerization activity / Amyloid fiber formation / positive regulation of transcription by RNA polymerase II / protein-containing complex / DNA binding / extracellular exosome / extracellular region / nucleoplasm / nucleus
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 H2A / Histone 2A ...: / 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 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 H3 signature 1. / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Histone-fold
Similarity search - Domain/homology
Histone H2B 1.1 / Histone H2A.Z / Histone H4 / Histone H3.3
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Homo sapiens (human) / Xenopus laevis (African clawed frog) / Mus musculus (house mouse) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsTan D / Sokolova V
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM133611 United States
National Science Foundation (NSF, United States)1942049 United States
CitationJournal: Epigenomes / Year: 2024
Title: Structural and Biochemical Characterization of the Nucleosome Containing Variants H3.3 and H2A.Z.
Authors: Harry Jung / Vladyslava Sokolova / Gahyun Lee / Victoria Rose Stevens / Dongyan Tan /
Abstract: Variant H3.3, along with H2A.Z, is notably enriched at promoter regions and is commonly associated with transcriptional activation. However, the specific molecular mechanisms through which H3.3 ...Variant H3.3, along with H2A.Z, is notably enriched at promoter regions and is commonly associated with transcriptional activation. However, the specific molecular mechanisms through which H3.3 influences chromatin dynamics at transcription start sites, and its role in gene regulation, remain elusive. Using a combination of biochemistry and cryo-electron microscopy (cryo-EM), we show that the inclusion of H3.3 alone does not induce discernible changes in nucleosome DNA dynamics. Conversely, the presence of both H3.3 and H2A.Z enhances DNA's flexibility similarly to H2A.Z alone. Interestingly, our findings suggest that the presence of H3.3 in the H2A.Z nucleosome provides slightly increased protection to DNA at internal sites within the nucleosome. These results imply that while H2A.Z at active promoters promotes the formation of more accessible nucleosomes with increased DNA accessibility to facilitate transcription, the simultaneous presence of H3.3 offers an additional mechanism to fine-tune nucleosome accessibility and the chromatin environment.
History
DepositionMar 19, 2024-
Header (metadata) releaseJun 12, 2024-
Map releaseJun 12, 2024-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44148.png

Downloads & links

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Map

FileDownload / File: emd_44148.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.)
1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.10620304 - 0.22206807
Average (Standard dev.)0.00021574917 (±0.004335793)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_44148_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_44148_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : nucleosome containing histone variants H3.3 and H2A.Z

EntireName: nucleosome containing histone variants H3.3 and H2A.Z
Components
  • Complex: nucleosome containing histone variants H3.3 and H2A.Z
    • Protein or peptide: Histone H3.3
    • Protein or peptide: Histone H4
    • Protein or peptide: Histone H2A.Z
    • Protein or peptide: Histone H2B 1.1
    • DNA: DNA (128-MER)
    • DNA: DNA (128-MER)

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Supramolecule #1: nucleosome containing histone variants H3.3 and H2A.Z

SupramoleculeName: nucleosome containing histone variants H3.3 and H2A.Z / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: The complex contains Xenopus histone H2B and H4, human H3.3, and mouse H2A.Z
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 288 KDa

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Macromolecule #1: Histone H3.3

MacromoleculeName: Histone H3.3 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 15.360983 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MARTKQTARK STGGKAPRKQ LATKAARKSA PSTGGVKKPH RYRPGTVALR EIRRYQKSTE LLIRKLPFQR LVREIAQDFK TDLRFQSAA IGALQEASEA YLVGLFEDTN LCAIHAKRVT IMPKDIQLAR RIRGERA

UniProtKB: Histone H3.3

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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: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 11.394426 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSGRGKGGKG LGKGGAKRHR KVLRDNIQGI TKPAIRRLAR RGGVKRISGL IYEETRGVLK VFLENVIRDA VTYTEHAKRK TVTAMDVVY ALKRQGRTLY GFGG

UniProtKB: Histone H4

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Macromolecule #3: Histone H2A.Z

MacromoleculeName: Histone H2A.Z / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 13.581796 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MAGGKAGKDS GKAKTKAVSR SQRAGLQFPV GRIHRHLKSR TTSHGRVGAT AAVYSAAILE YLTAEVLELA GNASKDLKVK RITPRHLQL AIRGDEELDS LIKATIAGGG VIPHIHKSLI GKKGQQKTV

UniProtKB: Histone H2A.Z

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Macromolecule #4: Histone H2B 1.1

MacromoleculeName: Histone H2B 1.1 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 13.965265 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MPEPAKSAPA PKKGSKKAVT KTQKKDGKKR RKSRKESYAI YVYKVLKQVH PDTGISSKAM SIMNSFVNDV FERIAGEASR LAHYNKRST ITSREIQTAV RLLLPGELAK HAVSEGTKAV TKYTSAK

UniProtKB: Histone H2B 1.1

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Macromolecule #5: DNA (128-MER)

MacromoleculeName: DNA (128-MER) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 45.604047 KDa
SequenceString: (DA)(DC)(DA)(DG)(DG)(DA)(DT)(DG)(DT)(DA) (DT)(DA)(DT)(DA)(DT)(DC)(DT)(DG)(DA)(DC) (DA)(DC)(DG)(DT)(DG)(DC)(DC)(DT)(DG) (DG)(DA)(DG)(DA)(DC)(DT)(DA)(DG)(DG)(DG) (DA) (DG)(DT)(DA)(DA)(DT)(DC) ...String:
(DA)(DC)(DA)(DG)(DG)(DA)(DT)(DG)(DT)(DA) (DT)(DA)(DT)(DA)(DT)(DC)(DT)(DG)(DA)(DC) (DA)(DC)(DG)(DT)(DG)(DC)(DC)(DT)(DG) (DG)(DA)(DG)(DA)(DC)(DT)(DA)(DG)(DG)(DG) (DA) (DG)(DT)(DA)(DA)(DT)(DC)(DC)(DC) (DC)(DT)(DT)(DG)(DG)(DC)(DG)(DG)(DT)(DT) (DA)(DA) (DA)(DA)(DC)(DG)(DC)(DG)(DG) (DG)(DG)(DG)(DA)(DC)(DA)(DG)(DC)(DG)(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)(DC)(DT) (DC)(DC)(DA)(DG)

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Macromolecule #6: DNA (128-MER)

MacromoleculeName: DNA (128-MER) / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 51.269656 KDa
SequenceString: (DA)(DT)(DC)(DC)(DC)(DG)(DC)(DC)(DC)(DT) (DG)(DG)(DA)(DG)(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) ...String:
(DA)(DT)(DC)(DC)(DC)(DG)(DC)(DC)(DC)(DT) (DG)(DG)(DA)(DG)(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)(DC) (DG)(DC)(DT)(DG)(DT)(DC) (DC)(DC)(DC)(DC)(DG)(DC)(DG)(DT)(DT)(DT) (DT)(DA)(DA)(DC) (DC)(DG)(DC)(DC)(DA) (DA)(DG)(DG)(DG)(DG)(DA)(DT)(DT)(DA)(DC) (DT)(DC)(DC)(DC)(DT) (DA)(DG)(DT)(DC) (DT)(DC)(DC)(DA)(DG)(DG)(DC)(DA)(DC)(DG) (DT)(DG)(DT)(DC)(DA)(DG) (DA)(DT)(DA) (DT)(DA)(DT)(DA)(DC)(DA)(DT)(DC)(DC)(DT) (DG)(DT)(DG)(DC)(DA)(DT)(DG) (DA)(DC) (DT)(DA)(DG)(DA)(DT)

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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

Concentration0.8 mg/mL
BufferpH: 7.5 / Details: 20mM Tris-HCl, 5mM NaCl
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Pressure: 0.0002 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV
Details: Freezing condition: blot force 0, blot time 4.5 second.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 5140 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 81000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.25 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1291847
Startup modelType of model: INSILICO MODEL
In silico model: relion-4.0 was used to generate a de novo 3D initial model from selected 2D particles.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 205792
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final 3D classificationNumber classes: 4 / Avg.num./class: 320000 / Software - Name: RELION (ver. 4.0)

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Atomic model buiding 1

Initial model
PDB IDChain

5b33

chain_id: A, source_name: PDB, initial_model_type: experimental model

5b33

chain_id: B, source_name: PDB, initial_model_type: experimental model

5b33

chain_id: C, source_name: PDB, initial_model_type: experimental model

5b33

chain_id: D, source_name: PDB, initial_model_type: experimental model

5b33

chain_id: E, source_name: PDB, initial_model_type: experimental model

5b33

chain_id: F, source_name: PDB, initial_model_type: experimental model

5b33

chain_id: G, source_name: PDB, initial_model_type: experimental model

5b33

chain_id: H, source_name: PDB, initial_model_type: experimental model

6fq5

chain_id: I, source_name: PDB, initial_model_type: experimental model

6fq5

chain_id: J, source_name: PDB, initial_model_type: experimental model
DetailsPhenix real-space refinement was used.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 87.42
Output model

PDB-9b3p:
The cryo-EM structure of the H2A.Z-H3.3 double-variant nucleosome

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