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- EMDB-68024: Full-length Clr4 Binding to H3K14 Ubiquitinated Nucleosome -

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Basic information

Entry
Database: EMDB / ID: EMD-68024
TitleFull-length Clr4 Binding to H3K14 Ubiquitinated Nucleosome
Map data
Sample
  • Complex: Clr4-H3K9NleK14Ub nucleosome complex
    • Protein or peptide: Histone H2A
    • Protein or peptide: Histone H2B
    • Protein or peptide: Histone H3
    • Protein or peptide: Histone H2A
    • Protein or peptide: Clr4
    • Protein or peptide: Ubiquitin
    • DNA: Widom 601 DNA
    • DNA: Widom 601 DNA
KeywordsMethylation / Nucleosome / GENE REGULATION
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsDu YX / Ai HS
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32501108 China
CitationJournal: Angew Chem Int Ed Engl / Year: 2026
Title: Chemically Synthesized H3K14Ub Unveils Clr4's IDR-Mediated Multivalent Nucleosome Recognition in H3K9 Methylation.
Authors: Maoshen Sun / Yunxiang Du / Zhengqing Li / Akejiang Aderjiang / Meixuan Xin / Huasong Ai /
Abstract: Histone H3 lysine 9 methylation (H3K9me) is a central epigenetic mark governing heterochromatin formation. Although the H3K9 methyltransferase Clr4 has been extensively characterized using short ...Histone H3 lysine 9 methylation (H3K9me) is a central epigenetic mark governing heterochromatin formation. Although the H3K9 methyltransferase Clr4 has been extensively characterized using short histone peptide substrates, how it recognizes and coordinates different structural domains to engage physiological substrate nucleosomes remains poorly understood. Here, we employed chemical protein synthesis to generate site-specifically ubiquitinated H3K14Ub histones and nucleosomes, enabling quantitative biochemical and structural investigations. Using a CAET handle-assisted strategy, we obtained homogeneous H3K14Ub nucleosomes and demonstrated that ubiquitination enhances Clr4 activity by ∼350-fold on nucleosomes relative to unmodified substrates. Clr4 domain deletion analyses revealed that, the intrinsically disordered region (IDR) of Clr4 is critical for nucleosome binding and ubiquitin-dependent stimulation. Through site-directed photo-crosslinking, we identified specific IDR residues mediating interactions with nucleosomal surfaces. Furthermore, using an isoUb-based synthetic approach, we generated H3K9NleK14Ub nucleosomes and determined cryo-EM structures of Clr4-nucleosome complexes, unveiling multivalent nucleosome recognition by the IDR via four distinct interfaces: the H2A-H2B acidic patch, the H2A/H2B basic groove, the H2B and H3 elbow regions. Methyltransferase activity assays confirmed that mutations disrupting these interfaces impair Clr4 activity. Our study provides mechanistic insights into the ubiquitin-dependent activation mechanism of Clr4, highlighting the power of chemical biology in deciphering epigenetic regulation.
History
DepositionDec 29, 2025-
Header (metadata) releaseApr 22, 2026-
Map releaseApr 22, 2026-
UpdateApr 22, 2026-
Current statusApr 22, 2026Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_68024.png

Downloads & links

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Map

FileDownload / File: emd_68024.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
0.97 Å/pix.
x 384 pix.
= 372.48 Å		0.97 Å/pix.
x 384 pix.
= 372.48 Å		0.97 Å/pix.
x 384 pix.
= 372.48 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.97 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.6979824 - 1.8215631
Average (Standard dev.)0.0006480917 (±0.040879462)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 372.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_68024_additional_1.map
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Half map: #1

Fileemd_68024_half_map_1.map
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Half map: #2

Fileemd_68024_half_map_2.map
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Sample components

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Entire : Clr4-H3K9NleK14Ub nucleosome complex

EntireName: Clr4-H3K9NleK14Ub nucleosome complex
Components
  • Complex: Clr4-H3K9NleK14Ub nucleosome complex
    • Protein or peptide: Histone H2A
    • Protein or peptide: Histone H2B
    • Protein or peptide: Histone H3
    • Protein or peptide: Histone H2A
    • Protein or peptide: Clr4
    • Protein or peptide: Ubiquitin
    • DNA: Widom 601 DNA
    • DNA: Widom 601 DNA

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Supramolecule #1: Clr4-H3K9NleK14Ub nucleosome complex

SupramoleculeName: Clr4-H3K9NleK14Ub nucleosome complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 270 KDa

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Macromolecule #1: Histone H2A

MacromoleculeName: Histone H2A / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString:
SGRGKQGGKA RAKAKTRSSR AGLQFPVGRV HRLLRKGNYS ERVGAGAPVY LAAVLEYLTA EILELAGNAA RDNKKTRIIP RHLQLAIRND EELNKLLGRV TIAQGGVLPN IQAVLLPKKT ESHHKAKGK

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Macromolecule #2: Histone H2B

MacromoleculeName: Histone H2B / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString:
PEPAKSAPAP KKGSKKAVTK AQKKDGKKRK RSRKESYSVY VYKVLKQVHP DTGISSKAMG IMNSFVNDIF ERIAGEASRL AHYNKRSTIT SREIQTAVRL LLPGELAKHA VSEGTKAVTK YTSAK

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Macromolecule #3: Histone H3

MacromoleculeName: Histone H3 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString:
ARTKQTARKS TGGKAPRKQL ATKAARKSAP ATGGVKKPHR YRPGTVALRE IRRYQKSTEL LIRKLPFQRL VREIAQDFKT DLRFQSSAVM ALQEASEAYL VGLFEDTNLS AIHAKRVTIM PKDIQLARRI RGERA

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Macromolecule #4: Histone H2A

MacromoleculeName: Histone H2A / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString:
SGRGKGGKGL GKGGAKRHRK VLRDNIQGIT KPAIRRLARR GGVKRISGLI YEETRGVLKV FLENVIRDAV TYTEHAKRKT VTAMDVVYAL KRQGRTLYGF GG

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Macromolecule #5: Clr4

MacromoleculeName: Clr4 / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: MSPKQEEYEV ERIVDEKLDR NGAVKLYRIR WLNYSSRSDT WEPPENLSGC SAVLAEWKRR KRRLKGSNSD SDSPHHASNP HPNSRQKHQH QTSKSVPRSQ RFSRELNVKK ENKKVFSSQT TKRQSRKQST ALTTNDTSII LDDSLHTNSK KLGKTRNEVK EESQKRELVS ...String:
MSPKQEEYEV ERIVDEKLDR NGAVKLYRIR WLNYSSRSDT WEPPENLSGC SAVLAEWKRR KRRLKGSNSD SDSPHHASNP HPNSRQKHQH QTSKSVPRSQ RFSRELNVKK ENKKVFSSQT TKRQSRKQST ALTTNDTSII LDDSLHTNSK KLGKTRNEVK EESQKRELVS NSIKEATSPK TSSILTKPRN PSKLDSYTHL SFYEKRELFR KKLREIEGPE VTLVNEVDDE PCPSLDFQFI SQYRLTQGVI PPDPNFQSGC NCSSLGGCDL NNPSRCECLD DLDEPTHFAY DAQGRVRADT GAVIYECNSF CSCSMECPNR VVQRGRTLPL EIFKTKEKGW GVRSLRFAPA GTFITCYLGE VITSAEAAKR DKNYDDDGIT YLFDLDMFDD ASEYTVDAQN YGDVSRFFNH SCSPNIAIYS AVRNHGFRTI YDLAFFAIKD IQPLEELTFD YAGAKDFSPV QSQKSQQNRI SKLRRQCKCG SANCRGWLFG

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Macromolecule #6: Ubiquitin

MacromoleculeName: Ubiquitin / type: protein_or_peptide / ID: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString:
MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGG

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Macromolecule #7: Widom 601 DNA

MacromoleculeName: Widom 601 DNA / type: dna / ID: 7 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
SequenceString:
CTGGAGAATC CCGGTGCCGA GGCCGCTCAA TTGGTCGTAG ACAGCTCTAG CACCGCTTAA ACGCACGTAC GCGCTGTCCC CCGCGTTTTA ACCGCCAAGG GGATTACTCC CTAGTCTCCA GGCACGTGTC AGATATATAC ATCCTGT

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Macromolecule #8: Widom 601 DNA

MacromoleculeName: Widom 601 DNA / type: dna / ID: 8 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
SequenceString:
ACAGGATGTA TATATCTGAC ACGTGCCTGG AGACTAGGGA GTAATCCCCT TGGCGGTTAA AACGCGGGGG ACAGCGCGTA CGTGCGTTTA AGCGGTGCTA GAGCTGTCTA CGACCAATTG AGCGGCCTCG GCACCGGGAT TCTCCAG

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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: 7.2 / Details: 25mM HEPES, 100mM NaCl, 0.01mM ZnCl2, 0.01mM SAM
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware: (Name: Gctf, CTFFIND) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF
Software: (Name: cryoSPARC (ver. 3.2.0), RELION (ver. 3.1.1))
Number images used: 332014
Initial angle assignmentType: ANGULAR RECONSTITUTION
Software: (Name: RELION (ver. 3.1.1), cryoSPARC (ver. 3.2.0))
Final angle assignmentType: ANGULAR RECONSTITUTION
Software: (Name: RELION (ver. 3.1.1), cryoSPARC (ver. 3.2.0))
Final 3D classificationNumber classes: 1 / Avg.num./class: 332014
Software: (Name: RELION (ver. 3.1.1), cryoSPARC (ver. 3.2.0))
FSC plot (resolution estimation)

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