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- PDB-9w74: Cryo-EM structure of the close-packed di-hexasome (CPDH) -

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Basic information

Entry
Database: PDB / ID: 9w74
TitleCryo-EM structure of the close-packed di-hexasome (CPDH)
Components
  • (DNA (207-MER)) x 2
  • Histone H2A type 1
  • Histone H2B 1.1
  • Histone H3.2
  • Histone H4
KeywordsGENE REGULATION / Chromatin / Nucleosome / Hexasome / Subnucleosome / Histone / DNA
Function / homology
Function and homology information


structural constituent of chromatin / heterochromatin formation / nucleosome / nucleosome assembly / protein heterodimerization activity / DNA binding / 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 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 H3 signature 1. / 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 H2B 1.1 / Histone H2A type 1 / Histone H4 / Histone H3.2
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.94 Å
AuthorsHo, C.-H. / Takizawa, Y. / Kurumizaka, H.
Funding support Japan, 8items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP23H05475 Japan
Japan Society for the Promotion of Science (JSPS)JP24H02319 Japan
Japan Society for the Promotion of Science (JSPS)JP24H02328 Japan
Japan Science and TechnologyJPMJER1901 Japan
Japan Science and TechnologyJPMJCR24T3 Japan
Japan Agency for Medical Research and Development (AMED)JP25ama121009 Japan
Japan Agency for Medical Research and Development (AMED)JP25ama121002 Japan
Japan Society for the Promotion of Science (JSPS)JP25K18403 Japan
CitationJournal: iScience / Year: 2026
Title: A method for cryo-EM analysis of eukaryotic nucleosomes reconstituted in bacterial cells.
Authors: Cheng-Han Ho / Yuki Kobayashi / Mitsuo Ogasawara / Yoshimasa Takizawa / Hitoshi Kurumizaka /
Abstract: Conventional methods for preparing nucleosomes are time-consuming and technically demanding. In the present study, we extended the approach of generating nucleosomes in by the co-expression of all ...Conventional methods for preparing nucleosomes are time-consuming and technically demanding. In the present study, we extended the approach of generating nucleosomes in by the co-expression of all four histones, allowing nucleosomes to be assembled in cells. The bacterially reconstituted nucleosomes can be readily prepared from the cells and directly subjected to cryo-EM single particle analysis. Using this method, we obtained a 2.56 Å nucleosome structure that is highly similar to a previously reported nucleosome crystal structure, validating the use of nucleosomes formed in for cryo-EM analysis. Unexpectedly, we also discovered a non-canonical nucleosome structure, in which two hexasomes are closely packed. This system provides a robust and efficient platform for structural studies of nucleosomes and nucleosome variants, and may facilitate the discovery of chromatin architectures.
History
DepositionAug 5, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 10, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Histone H3.2
B: Histone H4
C: Histone H2A type 1
D: Histone H2B 1.1
E: Histone H3.2
F: Histone H4
K: Histone H3.2
L: Histone H4
M: Histone H2A type 1
N: Histone H2B 1.1
O: Histone H3.2
P: Histone H4
I: DNA (207-MER)
J: DNA (207-MER)


Theoretical massNumber of molelcules
Total (without water)306,16814
Polymers306,16814
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 12 molecules AEKOBFLPCMDN

#1: Protein
Histone H3.2


Mass: 15435.126 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Production host: Escherichia coli (E. coli) / References: UniProt: P84233
#2: Protein
Histone H4


Mass: 13307.514 Da / Num. of mol.: 4
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 type 1


Mass: 18086.951 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: P06897
#4: Protein Histone H2B 1.1 / H2B1.1


Mass: 13655.948 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 (207-MER)


Mass: 64788.910 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: The molecule was purified directly from E. coli cells, so it contains random E. coli genomic DNA. The poly-A sequence is used since it cannot be assigned the specific sequence
Source: (natural) Escherichia coli (E. coli)
#6: DNA chain DNA (207-MER)


Mass: 62922.816 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: The molecule was purified directly from E. coli cells, so it contains random E. coli genomic DNA. The poly-T sequence is used since it cannot be assigned the specific sequence
Source: (natural) Escherichia coli (E. coli)

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Details

Has protein modificationN

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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

Component
IDNameTypeEntity IDParent-IDSource
1Close-packed di-hexasome (CPDH) in complex with the PL2-6 scFvCOMPLEXall0RECOMBINANT
2Close-packed di-hexasome (CPDH)COMPLEXall1RECOMBINANT
3PL2-6 scFvCOMPLEX1RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Xenopus laevis (African clawed frog)8355
32Xenopus laevis (African clawed frog)8355
43Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32Escherichia coli (E. coli)562
43Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2250 nm / Nominal defocus min: 1250 nm
Image recordingElectron dose: 60.1 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.7.1particle selectionBlob picker
13cryoSPARC4.7.13D reconstructionNon-uniform Refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.94 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 4963 / Symmetry type: POINT
RefinementHighest resolution: 4.94 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00618412
ELECTRON MICROSCOPYf_angle_d0.76226636
ELECTRON MICROSCOPYf_dihedral_angle_d32.9436226
ELECTRON MICROSCOPYf_chiral_restr0.043032
ELECTRON MICROSCOPYf_plane_restr0.0061938

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