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- PDB-7otq: Cryo-EM structure of ALC1/CHD1L bound to a PARylated nucleosome -

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Basic information

Entry
Database: PDB / ID: 7otq
TitleCryo-EM structure of ALC1/CHD1L bound to a PARylated nucleosome
Components
  • (DNA (149-MER) Widom 601 ...) x 2
  • Chromodomain-helicase-DNA-binding protein 1-like
  • Histone H2A type 1
  • Histone H2B 1.1
  • Histone H3.2
  • Histone H4
KeywordsDNA BINDING PROTEIN / ALC1 / CHD1L / chromatin remodeler / DNA damage response / nucleosome / poly(ADP-ribose)
Function / homology
Function and homology information


poly-ADP-D-ribose modification-dependent protein binding / ATP-dependent chromatin remodeler activity / site of DNA damage / nucleosome binding / DNA helicase activity / histone reader activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / Dual Incision in GG-NER / Formation of Incision Complex in GG-NER / structural constituent of chromatin ...poly-ADP-D-ribose modification-dependent protein binding / ATP-dependent chromatin remodeler activity / site of DNA damage / nucleosome binding / DNA helicase activity / histone reader activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / Dual Incision in GG-NER / Formation of Incision Complex in GG-NER / structural constituent of chromatin / nucleosome / site of double-strand break / chromatin remodeling / protein heterodimerization activity / nucleotide binding / DNA repair / DNA damage response / ATP hydrolysis activity / DNA binding / nucleoplasm / ATP binding / nucleus / plasma membrane / cytosol
Similarity search - Function
Chromodomain-helicase-DNA-binding protein 1-like / : / SNF2-like, N-terminal domain superfamily / SNF2, N-terminal / SNF2-related domain / DNA/RNA helicase, ATP-dependent, DEAH-box type, conserved site / DEAH-box subfamily ATP-dependent helicases signature. / Histone H2B signature. / Histone H2B / Histone H2B ...Chromodomain-helicase-DNA-binding protein 1-like / : / SNF2-like, N-terminal domain superfamily / SNF2, N-terminal / SNF2-related domain / 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. / 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 / Helicase conserved C-terminal 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 / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / Macro domain / Macro domain profile. / Macro domain-like / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / Histone H2B 1.1 / Histone H2A type 1 / Histone H4 / Histone H3.2 / Chromodomain-helicase-DNA-binding protein 1-like
Similarity search - Component
Biological speciesHomo sapiens (human)
Xenopus laevis (African clawed frog)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsBacic, L. / Gaullier, G. / Deindl, S.
Funding support Sweden, 4items
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, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 15, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
K: Chromodomain-helicase-DNA-binding protein 1-like
A: Histone H3.2
B: Histone H4
C: Histone H2A type 1
D: Histone H2B 1.1
E: Histone H3.2
F: Histone H4
G: Histone H2A type 1
H: Histone H2B 1.1
I: DNA (149-MER) Widom 601 sequence
J: DNA (149-MER) Widom 601 sequence


Theoretical massNumber of molelcules
Total (without water)306,81411
Polymers306,81411
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area58280 Å2
ΔGint-389 kcal/mol
Surface area92760 Å2
MethodPISA

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Components

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Protein , 5 types, 9 molecules KAEBFCGDH

#1: Protein Chromodomain-helicase-DNA-binding protein 1-like / Amplified in liver cancer protein 1


Mass: 98906.766 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: C-terminal 6-His tag. / Source: (gene. exp.) Homo sapiens (human) / Gene: CHD1L, ALC1 / Plasmid: pNIC-CH2 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta 2 / References: UniProt: Q86WJ1, DNA helicase
#2: Protein Histone H3.2


Mass: 15403.062 Da / Num. of mol.: 2 / Mutation: C110A
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
#3: Protein Histone H4 /


Mass: 11394.426 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: P62799
#4: Protein Histone H2A type 1


Mass: 14109.436 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
#5: 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 (149-MER) Widom 601 ... , 2 types, 2 molecules IJ

#6: DNA chain DNA (149-MER) Widom 601 sequence


Mass: 49175.336 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#7: DNA chain DNA (149-MER) Widom 601 sequence


Mass: 49606.586 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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

ComponentName: ALC1/CHD1L bound to a PARylated nucleosome / Type: COMPLEX
Details: The nucleosome was PARylated by PARP2 and HPF1 before addition of ALC1.
Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.305 MDa / Experimental value: NO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Buffer solutionpH: 7.5
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Current 20 mA. / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
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
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.2 sec. / Electron dose: 45 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 26747 / Details: Total dose was fractionated over 40 movie frames.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
2EPU2.6.1image acquisition
4CTFFIND4.1.9CTF correction
7ISOLDE1model fitting
9RELION3.1.1initial Euler assignment
10cryoSPARC3.2.0final Euler assignment
11cryoSPARC3.2.0classification
12cryoSPARC3.2.03D reconstruction
13PHENIX1.19.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3321590
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 5487 / Algorithm: FOURIER SPACE / Details: Non-uniform refinement in cryoSPARC 3.2.0. / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Real-space CC
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
16RYR

6ryr

A1
26JYL

6jyl

K1
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 269.45 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004216671
ELECTRON MICROSCOPYf_angle_d0.75723793
ELECTRON MICROSCOPYf_chiral_restr0.04822700
ELECTRON MICROSCOPYf_plane_restr0.01221989
ELECTRON MICROSCOPYf_dihedral_angle_d24.90956729

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