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- EMDB-13065: Cryo-EM structure of ALC1/CHD1L bound to a PARylated nucleosome -

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Basic information

Entry
Database: EMDB / ID: EMD-13065
TitleCryo-EM structure of ALC1/CHD1L bound to a PARylated nucleosome
Map data
SampleALC1/CHD1L bound to a PARylated nucleosome
  • Chromodomain-helicase-DNA-binding protein 1-like
  • Histone H3.2
  • Histone H4
  • Histone H2A type 1
  • Histone H2B 1.1
  • (nucleic-acidNucleic acid) x 2
Function / homology
Function and homology information


ATP-dependent chromatin remodeler activity => GO:0140658 / nucleotide-excision repair, preincision complex stabilization / nucleotide-excision repair, DNA incision, 3'-to lesion / nucleotide-excision repair, DNA duplex unwinding / global genome nucleotide-excision repair / nucleotide-excision repair, preincision complex assembly / DNA-templated transcription, initiation / nucleotide-excision repair, DNA incision, 5'-to lesion / Dual Incision in GG-NER / nucleotide-excision repair, DNA incision ...ATP-dependent chromatin remodeler activity => GO:0140658 / nucleotide-excision repair, preincision complex stabilization / nucleotide-excision repair, DNA incision, 3'-to lesion / nucleotide-excision repair, DNA duplex unwinding / global genome nucleotide-excision repair / nucleotide-excision repair, preincision complex assembly / DNA-templated transcription, initiation / nucleotide-excision repair, DNA incision, 5'-to lesion / Dual Incision in GG-NER / nucleotide-excision repair, DNA incision / Formation of Incision Complex in GG-NER / nucleosome / DNA helicase / DNA helicase activity / chromatin remodeling / ATP hydrolysis activity / protein heterodimerization activity / DNA repair / cellular response to DNA damage stimulus / nucleotide binding / DNA binding / nucleoplasm / ATP binding / plasma membrane / nucleus / cytosol
Similarity search - Function
Chromodomain-helicase-DNA-binding protein 1-like / SNF2-like, N-terminal domain superfamily / SNF2-related domain / SNF2, N-terminal / DNA/RNA helicase, ATP-dependent, DEAH-box type, conserved site / DEAH-box subfamily ATP-dependent helicases signature. / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site ...Chromodomain-helicase-DNA-binding protein 1-like / SNF2-like, N-terminal domain superfamily / SNF2-related domain / SNF2, N-terminal / DNA/RNA helicase, ATP-dependent, DEAH-box type, conserved site / DEAH-box subfamily ATP-dependent helicases signature. / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / C-terminus of histone H2A / Histone H2A, C-terminal domain / Histone 2A / Histone H2A / Histone H4 signature. / Histone H4, conserved site / 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 H3 signature 1. / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Helicase conserved C-terminal domain / Core histone H2A/H2B/H3/H4 / Histone H2A/H2B/H3 / Macro domain / Macro domain profile. / Histone-fold / Macro domain-like / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Helicase, C-terminal / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Histone H2B 1.1 / Histone H2A type 1 / Histone H4 / Histone H3.2 / Chromodomain-helicase-DNA-binding protein 1-like
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog) / Homo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsBacic L / Gaullier G / Deindl S
Funding support Sweden, 4 items
OrganizationGrant numberCountry
European Research Council (ERC)714068 Sweden
Knut and Alice Wallenberg Foundation019.0306 Sweden
Swedish Research Council2019-03534 Sweden
Cancerfonden19 0055 Pj Sweden
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
DepositionJun 10, 2021-
Header (metadata) releaseSep 15, 2021-
Map releaseSep 15, 2021-
UpdateSep 15, 2021-
Current statusSep 15, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.15
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.15
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7otq
  • Surface level: 0.15
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_13065.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 400 pix.
= 430.08 Å
1.08 Å/pix.
x 400 pix.
= 430.08 Å
1.08 Å/pix.
x 400 pix.
= 430.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0752 Å
Density
Contour LevelBy AUTHOR: 0.15 / Movie #1: 0.15
Minimum - Maximum-0.39473256 - 0.8218156
Average (Standard dev.)7.375511e-05 (±0.022399737)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 430.08 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.07521.07521.0752
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z430.080430.080430.080
α/β/γ90.00090.00090.000
start NX/NY/NZ1331310
NX/NY/NZ223226424
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.3950.8220.000

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Supplemental data

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Segmentation: #1

Fileemd_13065_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map 2

Fileemd_13065_half_map_1.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map 1

Fileemd_13065_half_map_2.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire ALC1/CHD1L bound to a PARylated nucleosome

EntireName: ALC1/CHD1L bound to a PARylated nucleosome
Details: The nucleosome was PARylated by PARP2 and HPF1 before addition of ALC1.
Number of Components: 8

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Component #1: protein, ALC1/CHD1L bound to a PARylated nucleosome

ProteinName: ALC1/CHD1L bound to a PARylated nucleosome
Details: The nucleosome was PARylated by PARP2 and HPF1 before addition of ALC1.
Recombinant expression: No
MassTheoretical: 305 kDa
SourceSpecies: Xenopus laevis (African clawed frog)

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Component #2: protein, Chromodomain-helicase-DNA-binding protein 1-like

ProteinName: Chromodomain-helicase-DNA-binding protein 1-like / Details: C-terminal 6-His tag. / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 98.906766 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, Histone H3.2

ProteinName: Histone H3.2 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 15.403062 kDa
SourceSpecies: Xenopus laevis (African clawed frog)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, Histone H4

ProteinName: Histone H4 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 11.394426 kDa
SourceSpecies: Xenopus laevis (African clawed frog)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #5: protein, Histone H2A type 1

ProteinName: Histone H2A type 1 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 14.109436 kDa
SourceSpecies: Xenopus laevis (African clawed frog)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: protein, Histone H2B 1.1

ProteinName: Histone H2B 1.1 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 13.655948 kDa
SourceSpecies: Xenopus laevis (African clawed frog)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #7: nucleic-acid, DNA (149-MER) Widom 601 sequence

nucleic acidName: DNA (149-MER) Widom 601 sequence / Class: DNA / Structure: OTHER / Synthetic: No
Sequence: (DT)(DC)(DT)(DA)(DG)(DG)(DT)(DG)(DA)(DC) (DC)(DA)(DT)(DC)(DA)(DG)(DA)(DA)(DT)(DC) (DC)(DC)(DG)(DG)(DT)(DG)(DC)(DC)(DG)(DA) (DG)(DG)(DC)(DC)(DG)(DC)(DT)(DC)(DA)(DA) (DT)(DT)(DG)(DG)(DT)(DC) ...Sequence:
(DT)(DC)(DT)(DA)(DG)(DG)(DT)(DG)(DA)(DC) (DC)(DA)(DT)(DC)(DA)(DG)(DA)(DA)(DT)(DC) (DC)(DC)(DG)(DG)(DT)(DG)(DC)(DC)(DG)(DA) (DG)(DG)(DC)(DC)(DG)(DC)(DT)(DC)(DA)(DA) (DT)(DT)(DG)(DG)(DT)(DC)(DG)(DT)(DA)(DG) (DA)(DC)(DA)(DG)(DC)(DT)(DC)(DT)(DA)(DG) (DC)(DA)(DC)(DC)(DG)(DC)(DT)(DT)(DA)(DA) (DA)(DC)(DG)(DC)(DA)(DC)(DG)(DT)(DA)(DC) (DG)(DC)(DG)(DC)(DT)(DG)(DT)(DC)(DC)(DC) (DC)(DC)(DG)(DC)(DG)(DT)(DT)(DT)(DT)(DA) (DA)(DC)(DC)(DG)(DC)(DC)(DA)(DA)(DG)(DG) (DG)(DG)(DA)(DT)(DT)(DA)(DC)(DT)(DC)(DC) (DC)(DT)(DA)(DG)(DT)(DC)(DT)(DC)(DC)(DA) (DG)(DG)(DC)(DA)(DC)(DG)(DT)(DG)(DT)(DC) (DA)(DG)(DA)(DT)(DA)(DT)(DA)(DT)(DA)(DC) (DA)(DT)(DC)(DG)(DA)(DT)(DA)(DG)(DG)(DC)
MassTheoretical: 49.175336 kDa
SourceSpecies: synthetic construct (others)

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Component #8: nucleic-acid, DNA (149-MER) Widom 601 sequence

nucleic acidName: DNA (149-MER) Widom 601 sequence / Class: DNA / Structure: OTHER / Synthetic: No
Sequence: (DG)(DC)(DC)(DT)(DA)(DT)(DC)(DG)(DA)(DT) (DG)(DT)(DA)(DT)(DA)(DT)(DA)(DT)(DC)(DT) (DG)(DA)(DC)(DA)(DC)(DG)(DT)(DG)(DC)(DC) (DT)(DG)(DG)(DA)(DG)(DA)(DC)(DT)(DA)(DG) (DG)(DG)(DA)(DG)(DT)(DA) ...Sequence:
(DG)(DC)(DC)(DT)(DA)(DT)(DC)(DG)(DA)(DT) (DG)(DT)(DA)(DT)(DA)(DT)(DA)(DT)(DC)(DT) (DG)(DA)(DC)(DA)(DC)(DG)(DT)(DG)(DC)(DC) (DT)(DG)(DG)(DA)(DG)(DA)(DC)(DT)(DA)(DG) (DG)(DG)(DA)(DG)(DT)(DA)(DA)(DT)(DC)(DC) (DC)(DC)(DT)(DT)(DG)(DG)(DC)(DG)(DG)(DT) (DT)(DA)(DA)(DA)(DA)(DC)(DG)(DC)(DG)(DG) (DG)(DG)(DG)(DA)(DC)(DA)(DG)(DC)(DG)(DC) (DG)(DT)(DA)(DC)(DG)(DT)(DG)(DC)(DG)(DT) (DT)(DT)(DA)(DA)(DG)(DC)(DG)(DG)(DT)(DG) (DC)(DT)(DA)(DG)(DA)(DG)(DC)(DT)(DG)(DT) (DC)(DT)(DA)(DC)(DG)(DA)(DC)(DC)(DA)(DA) (DT)(DT)(DG)(DA)(DG)(DC)(DG)(DG)(DC)(DC) (DT)(DC)(DG)(DG)(DC)(DA)(DC)(DC)(DG)(DG) (DG)(DA)(DT)(DT)(DC)(DT)(DG)(DA)(DT)(DG) (DG)(DT)(DC)(DA)(DC)(DC)(DT)(DA)(DG)(DA)
MassTheoretical: 49.606586 kDa
SourceSpecies: synthetic construct (others)

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Experimental details

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Sample preparation

SpecimenSpecimen State: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.3 mg/mL / pH: 7.5
Support filmCurrent 20 mA.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen Name: ETHANE / Temperature: 277 K / Humidity: 100 %
Details: 3 uL were applied on grid and immediately blotted for 2.5 s at blot force 0..

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: TFS KRIOS
Electron gunElectron Source: FIELD EMISSION GUN / Accelerating Voltage: 300 kV / Electron Dose: 45 e/Å2 / Illumination Mode: FLOOD BEAM
LensImaging Mode: BRIGHT FIELD / Energy Filter: GIF Bioquantum
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: OTHER

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Image acquisition

Image acquisitionNumber of Digital Images: 26747 / Details: Total dose was fractionated over 40 movie frames.

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Image processing

ProcessingMethod: single particle reconstruction / Applied Symmetry: C1 (asymmetric) / Number of Projections: 5487
3D reconstructionAlgorithm: FOURIER SPACE / Software: cryoSPARC / Resolution: 4.8 Å / Resolution Method: FSC 0.143 CUT-OFF / Details: Non-uniform refinement in cryoSPARC 3.2.0. / Euler angles: Non-uniform refinement in cryoSPARC.
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Target Criteria: Real-space CC / Refinement space: REAL
Input PDB model: 6RYR, 6JYL
Chain ID: A, K
Output model

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