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- EMDB-17944: Cryo-EM structure of an asymmetric nucleosome (10-N-0) from a Gla... -

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Basic information

Entry
Database: EMDB / ID: EMD-17944
TitleCryo-EM structure of an asymmetric nucleosome (10-N-0) from a Glacios/Falcon3EC
Map dataMap from homogeneous refinement in cryosparc.
Sample
  • Complex: 601 nucleosome with 10 additional bp
    • Protein or peptide: Histone H3.2
    • Protein or peptide: Histone H4
    • Protein or peptide: Histone H2A type 1
    • Protein or peptide: Histone H2B 1.1
    • DNA: Widom 601 sequence with 10 additional bp on the strong side
    • DNA: Widom 601 sequence with 10 additional bp on the strong side
KeywordsNucleosome / NCP / DNA / Histones / DNA BINDING PROTEIN
Function / homology
Function and homology information


structural constituent of chromatin / nucleosome / protein heterodimerization activity / DNA binding / 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 H2A / Histone 2A / Histone H4, conserved site ...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 H2A / Histone 2A / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / 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
Histone H2B 1.1 / Histone H2A type 1 / Histone H4 / Histone H3.2
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsGaullier G
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Elife / Year: 2021
Title: Structure and dynamics of the chromatin remodeler ALC1 bound to a PARylated nucleosome.
Authors: Luka Bacic / Guillaume Gaullier / Anton Sabantsev / Laura C Lehmann / Klaus Brackmann / Despoina Dimakou / Mario Halic / Graeme Hewitt / Simon J Boulton / Sebastian Deindl /
Abstract: The chromatin remodeler ALC1 is recruited to and activated by DNA damage-induced poly(ADP-ribose) (PAR) chains deposited by PARP1/PARP2/HPF1 upon detection of DNA lesions. ALC1 has emerged as a ...The chromatin remodeler ALC1 is recruited to and activated by DNA damage-induced poly(ADP-ribose) (PAR) chains deposited by PARP1/PARP2/HPF1 upon detection of DNA lesions. ALC1 has emerged as a candidate drug target for cancer therapy as its loss confers synthetic lethality in homologous recombination-deficient cells. However, structure-based drug design and molecular analysis of ALC1 have been hindered by the requirement for PARylation and the highly heterogeneous nature of this post-translational modification. Here, we reconstituted an ALC1 and PARylated nucleosome complex modified in vitro using PARP2 and HPF1. This complex was amenable to cryo-EM structure determination without cross-linking, which enabled visualization of several intermediate states of ALC1 from the recognition of the PARylated nucleosome to the tight binding and activation of the remodeler. Functional biochemical assays with PARylated nucleosomes highlight the importance of nucleosomal epitopes for productive remodeling and suggest that ALC1 preferentially slides nucleosomes away from DNA breaks.
History
DepositionJul 17, 2023-
Header (metadata) releaseJul 26, 2023-
Map releaseJul 26, 2023-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17944.png

Downloads & links

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Map

FileDownload / File: emd_17944.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap from homogeneous refinement in cryosparc.
Voxel sizeX=Y=Z: 0.952 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.15872094 - 0.6360682
Average (Standard dev.)0.0016911587 (±0.03464744)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 243.712 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17944_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Mask #2

Fileemd_17944_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Map after post-processing with deepEMhancer, using the "tighTarget"...

Fileemd_17944_additional_1.map
AnnotationMap after post-processing with deepEMhancer, using the "tighTarget" weights.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map A from homogeneous refinement in cryosparc.

Fileemd_17944_half_map_1.map
AnnotationHalf-map A from homogeneous refinement in cryosparc.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map B from homogeneous refinement in cryosparc.

Fileemd_17944_half_map_2.map
AnnotationHalf-map B from homogeneous refinement in cryosparc.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : 601 nucleosome with 10 additional bp

EntireName: 601 nucleosome with 10 additional bp
Components
  • Complex: 601 nucleosome with 10 additional bp
    • Protein or peptide: Histone H3.2
    • Protein or peptide: Histone H4
    • Protein or peptide: Histone H2A type 1
    • Protein or peptide: Histone H2B 1.1
    • DNA: Widom 601 sequence with 10 additional bp on the strong side
    • DNA: Widom 601 sequence with 10 additional bp on the strong side

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Supramolecule #1: 601 nucleosome with 10 additional bp

SupramoleculeName: 601 nucleosome with 10 additional bp / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: The histones were recombinantly expressed and purified. The DNA was prepared by PCR. The nucleosomes were assembled by salt gradient dialysis.
Molecular weightTheoretical: 205.8 KDa

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Macromolecule #1: Histone H3.2

MacromoleculeName: Histone H3.2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MARTKQTARK STGGKAPRKQ LATKAARKSA PATGGVKKPH RYRPGTVALR EIRRYQKSTE LLIRKLPFQR LVREIAQDFK TDLRFQSSAV MALQEASEAY LVALFEDTNL AAIHAKRVTI MPKDIQLARR IRGERA

UniProtKB: Histone H3.2

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Macromolecule #2: Histone H4

MacromoleculeName: Histone H4 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSGRGKGGKG LGKGGAKRHR KVLRDNIQGI TKPAIRRLAR RGGVKRISGL IYEETRGVLK VFLENVIRDA VTYTEHAKRK TVTAMDVVYA LKRQGRTLYG FGG

UniProtKB: Histone H4

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Macromolecule #3: Histone H2A type 1

MacromoleculeName: Histone H2A type 1 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSGRGKQGGK TRAKAKTRSS RAGLQFPVGR VHRLLRKGNY AERVGAGAPV YLAAVLEYLT AEILELAGNA ARDNKKTRII PRHLQLAVRN DEELNKLLGR VTIAQGGVLP NIQSVLLPKK TESSKSAKSK

UniProtKB: Histone H2A type 1

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Macromolecule #4: Histone H2B 1.1

MacromoleculeName: Histone H2B 1.1 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MAKSAPAPKK GSKKAVTKTQ KKDGKKRRKT RKESYAIYVY KVLKQVHPDT GISSKAMSIM NSFVNDVFER IAGEASRLAH YNKRSTITSR EIQTAVRLLL PGELAKHAVS EGTKAVTKYT SAK

UniProtKB: Histone H2B 1.1

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Macromolecule #5: Widom 601 sequence with 10 additional bp on the strong side

MacromoleculeName: Widom 601 sequence with 10 additional bp on the strong side
type: dna / ID: 5 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
AATCGATGTA TATATCTGAC ACGTGCCTGG AGACTAGGGA GTAATCCCCT TGGCGGTTAA AACGCGGGGG ACAGCGCGTA CGTGCGTTTA AGCGGTGCTA GAGCTGTCTA CGACCAATTG AGCGGCCTCG GCACCGGGAT TCTGATGGTC ACCTAGA

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Macromolecule #6: Widom 601 sequence with 10 additional bp on the strong side

MacromoleculeName: Widom 601 sequence with 10 additional bp on the strong side
type: dna / ID: 6 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
TCTAGGTGAC CATCAGAATC CCGGTGCCGA GGCCGCTCAA TTGGTCGTAG ACAGCTCTAG CACCGCTTAA ACGCACGTAC GCGCTGTCCC CCGCGTTTTA ACCGCCAAGG GGATTACTCC CTAGTCTCCA GGCACGTGTC AGATATATAC ATCGATT

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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

Concentration1.17 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTrisTris
0.1 mMEDTAEthylenediaminetetraacetic acidEDTAEthylenediaminetetraacetic acid
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.04 kPa / Details: Pelco easiGlow operated with a 30 mA current.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Blot time 4s Blot force 0 4 uL of sample were applied on the grid (2 uL on each side of the grid).
DetailsAt the time of vitrification, the nucleosome sample used had spent about one year stored at 4C (this increases the propensity of nucleosomes to unwrap upon vitrification).

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1537 / Average electron dose: 40.01 e/Å2 / Details: 40 frames per movie

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Image processing

Particle selectionNumber selected: 907065
Startup modelType of model: INSILICO MODEL
In silico model: Output of ab initio reconstruction in cryosparc.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.2.1+230427)
Final 3D classificationNumber classes: 6 / Software - Name: cryoSPARC (ver. v4.2.1+230427)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.2.1+230427)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.2.1+230427) / Number images used: 68401
FSC plot (resolution estimation)

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