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

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Basic information

Entry
Database: PDB / ID: 9b3p
TitleThe cryo-EM structure of the H2A.Z-H3.3 double-variant nucleosome
Components
  • (DNA (128-MER)) x 2
  • Histone H2A.Z
  • Histone H2B 1.1
  • Histone H3.3
  • Histone H4
KeywordsSTRUCTURAL PROTEIN/DNA / histone 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 / nucleus organization / spermatid development / subtelomeric heterochromatin formation ...negative regulation of chromosome condensation / Barr body / regulation of centromere complex assembly / pericentric heterochromatin formation / inner kinetochore / muscle cell differentiation / oocyte maturation / nucleus organization / spermatid development / subtelomeric heterochromatin formation / single fertilization / RNA polymerase II core promoter sequence-specific DNA binding / heterochromatin / Replacement of protamines by nucleosomes in the male pronucleus / nucleosomal DNA binding / Inhibition of DNA recombination at telomere / telomere organization / embryo implantation / 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 / SIRT1 negatively regulates rRNA expression / Chromatin modifications during the maternal to zygotic transition (MZT) / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / PRC2 methylates histones and DNA / Regulation of endogenous retroelements by KRAB-ZFP proteins / Defective pyroptosis / cellular response to estradiol stimulus / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / RNA Polymerase I Promoter Escape / Transcriptional regulation by small RNAs / Formation of the beta-catenin:TCF transactivating complex / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / NoRC negatively regulates rRNA expression / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / euchromatin / B-WICH complex positively regulates rRNA expression / multicellular organism growth / heterochromatin formation / Meiotic recombination / Pre-NOTCH Transcription and Translation / cellular response to insulin stimulus / Activation of anterior HOX genes in hindbrain development during early embryogenesis / Transcriptional regulation of granulopoiesis / osteoblast differentiation / male gonad development / structural constituent of chromatin / nucleosome / nucleosome assembly / chromatin organization / RUNX1 regulates transcription of genes involved in differentiation of HSCs / Factors involved in megakaryocyte development and platelet production / Senescence-Associated Secretory Phenotype (SASP) / positive regulation of cell growth / Oxidative Stress Induced Senescence / Estrogen-dependent gene expression / cell population proliferation / chromosome, telomeric region / protein heterodimerization activity / RNA polymerase II cis-regulatory region sequence-specific DNA binding / 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 H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone H4, conserved site / Histone H4 signature. / Histone H4 ...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 H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / Histone H2A / Histone 2A / 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 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
DNA / DNA (> 10) / DNA (> 100) / Histone H2B 1.1 / Histone H2A.Z / Histone H4 / Histone H3.3
Similarity search - Component
Biological speciesHomo sapiens (human)
Xenopus laevis (African clawed frog)
Mus musculus (house mouse)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsTan, D. / Sokolova, V.
Funding support United States, 2items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 12, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_id_ISSN / _citation.journal_volume ..._citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.2Jul 17, 2024Group: Data collection / Refinement description
Category: em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.initial_refinement_model_id / _em_admin.last_update ..._em_3d_fitting_list.initial_refinement_model_id / _em_admin.last_update / _pdbx_initial_refinement_model.accession_code / _pdbx_initial_refinement_model.source_name / _pdbx_initial_refinement_model.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Histone H3.3
B: Histone H4
C: Histone H2A.Z
D: Histone H2B 1.1
E: Histone H3.3
F: Histone H4
G: Histone H2A.Z
H: Histone H2B 1.1
I: DNA (128-MER)
J: DNA (128-MER)


Theoretical massNumber of molelcules
Total (without water)205,47910
Polymers205,47910
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, 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 H3.3


Mass: 15360.983 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: H3-3A, H3.3A, H3F3, H3F3A, PP781, H3-3B, H3.3B, H3F3B / Production host: Escherichia coli (E. coli) / References: UniProt: P84243
#2: Protein Histone H4


Mass: 11394.426 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Production host: Escherichia coli (E. coli) / References: UniProt: P62799
#3: Protein Histone H2A.Z / H2A/z


Mass: 13581.796 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: H2az1, H2afz, H2az / Production host: Escherichia coli (E. coli) / References: UniProt: P0C0S6
#4: Protein Histone H2B 1.1 / H2B1.1


Mass: 13965.265 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Production host: Escherichia coli (E. coli) / References: UniProt: P02281

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain DNA (128-MER)


Mass: 45604.047 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)
#6: DNA chain DNA (128-MER)


Mass: 51269.656 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)

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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: nucleosome containing histone variants H3.3 and H2A.Z / Type: COMPLEX
Details: The complex contains Xenopus histone H2B and H4, human H3.3, and mouse H2A.Z
Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.288 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5 / Details: 20mM Tris-HCl, 5mM NaCl
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K
Details: Freezing condition: blot force 0, blot time 4.5 second

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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 / Calibrated magnification: 81000 X / Nominal defocus max: 2250 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 5140
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategoryDetails
1RELION4particle selectiontemplate-based automatic particle picking was perform using selected 2D class averages as references.
2EPUimage acquisition
4CTFFIND4.1CTF correction
7UCSF ChimeraX1.5model fitting
9RELION4initial Euler assignment
10RELION4final Euler assignment
11RELION4classification
12RELION43D reconstruction
13PHENIX1.15.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1291847
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 205792 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 87.42 / Protocol: RIGID BODY FIT / Space: REAL / Details: Phenix real-space refinement was used.
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeChain-IDInitial refinement model-ID
15B33

5b33

A5B33A1
25B33

5b33

B5B33B1
35B33

5b33

C5B33C1
45B33

5b33

D5B33D1
55B33

5b33

E5B33E1
65B33

5b33

F5B33F1
75B33

5b33

G5B33G1
85B33

5b33

H5B33H1
96fq5

6fq5

I6fq5I
106fq5

6fq5

J6fq5J

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