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- EMDB-75080: Nucleosome with an SSB at SHL -2.8 in complex with the WGR domain... -

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Basic information

Entry
Database: EMDB / ID: EMD-75080
TitleNucleosome with an SSB at SHL -2.8 in complex with the WGR domain of human PARP2, Class 2, Focused map
Map data
Sample
  • Complex: Nucleosome with an SSB at SHL -2.8 in complex with the WGR domain of human PARP2
KeywordsDNA damage binding protein / DNA BINDING PROTEIN
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.09 Å
AuthorsKim TH / Jayathilake C / Virk RK / Gregory-Lott ER
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM154859 United States
American Cancer SocietyIRG-16-186-21 United States
CitationJournal: J Mol Biol / Year: 2026
Title: High-Yield Production of Modified DNA Enables Structural Analysis of PARP2 Recognition of Nucleosomal Single-Strand Breaks.
Authors: Chathuni Jayathilake / Clare E Mewhinney / Emily R Gregory-Lott / Rajbinder K Virk / Riya Nair / Junseo Yang / Eun Cho / Alexander G Day / Derek J Taylor / Tae Hun Kim /
Abstract: Preparation of high-quality nucleosomal DNA substrates in milligram quantities remains a major bottleneck for mechanistic studies of chromatin-associated processes. Here, we present an optimized ...Preparation of high-quality nucleosomal DNA substrates in milligram quantities remains a major bottleneck for mechanistic studies of chromatin-associated processes. Here, we present an optimized large-scale PCR workflow that enables rapid, low-cost production of diverse nucleosomal DNAs suitable for biochemical assays and high-resolution cryo-EM. Systematic optimization of amplification conditions yields milligram quantities of homogeneous DNA that can be fluorescently or biotin-labeled and enzymatically modified to introduce site-specific single-strand breaks (SSBs) or epigenetic marks. We also engineered an improved Nt.BsmAI nickase variant (R386D) that minimizes undesired double-strand cleavage while maintaining robust nicking activity. Using nucleosomes reconstituted with these engineered DNAs, we demonstrate the versatility of this platform across EMSA, biolayer interferometry, and cryo-EM. Structural analysis reveals how the PARP2 WGR domain engages an SSB within the nucleosome and uncovers associated shifts in H2B tail conformation that facilitate access to lesions positioned near the tail. Overall, this workflow provides a robust and scalable method for generating precisely modified nucleosomal substrates, enabling quantitative and structural dissection of PARP2-mediated DNA damage recognition and the coupled histone H2B tail rearrangements that facilitate lesion accessibility in chromatin.
History
DepositionJan 13, 2026-
Header (metadata) releaseMar 25, 2026-
Map releaseMar 25, 2026-
UpdateApr 8, 2026-
Current statusApr 8, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_75080.png

Downloads & links

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Map

FileDownload / File: emd_75080.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
0.91 Å/pix.
x 300 pix.
= 273. Å		0.91 Å/pix.
x 300 pix.
= 273. Å		0.91 Å/pix.
x 300 pix.
= 273. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.91 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0254
Minimum - Maximum-0.11739038 - 0.18864635
Average (Standard dev.)-0.00004808938 (±0.0021245084)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 273.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_75080_half_map_1.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Half map: #2

Fileemd_75080_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Nucleosome with an SSB at SHL -2.8 in complex with the WGR domain...

EntireName: Nucleosome with an SSB at SHL -2.8 in complex with the WGR domain of human PARP2
Components
  • Complex: Nucleosome with an SSB at SHL -2.8 in complex with the WGR domain of human PARP2

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Supramolecule #1: Nucleosome with an SSB at SHL -2.8 in complex with the WGR domain...

SupramoleculeName: Nucleosome with an SSB at SHL -2.8 in complex with the WGR domain of human PARP2
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Drosophila melanogaster (fruit fly)

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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: 7.4
GridModel: Quantifoil R2/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 %

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Electron microscopy

MicroscopeTFS GLACIOS
SoftwareName: EPU
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 29.55 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 4.7.0) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL / In silico model: Alphafold model
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.09 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7.0) / Number images used: 51547
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.0)
FSC plot (resolution estimation)

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