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- PDB-7ux9: Arabidopsis DDM1 bound to nucleosome (H2A.W, H2B, H3.3, H4, with ... -

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Basic information

Entry
Database: PDB / ID: 7ux9
TitleArabidopsis DDM1 bound to nucleosome (H2A.W, H2B, H3.3, H4, with 147 bp DNA)
Components
  • ATP-dependent DNA helicase DDM1
  • DNA (antisense strand)
  • DNA (sense strand)
  • Histone H2B
  • Histone H3.3H3F3A
  • Histone H4
  • Probable histone H2A.7
KeywordsGENE REGULATION / Chromatin remodeler / Helicase / ATPase / Complex
Function / homology
Function and homology information


rDNA protrusion / DNA-mediated transformation / retrotransposition / heterochromatin organization / plasmodesma / DNA methylation-dependent heterochromatin formation / ATP-dependent chromatin remodeler activity / plastid / heterochromatin / pericentric heterochromatin ...rDNA protrusion / DNA-mediated transformation / retrotransposition / heterochromatin organization / plasmodesma / DNA methylation-dependent heterochromatin formation / ATP-dependent chromatin remodeler activity / plastid / heterochromatin / pericentric heterochromatin / heterochromatin formation / epigenetic regulation of gene expression / DNA helicase activity / structural constituent of chromatin / nucleosome / DNA helicase / chromatin remodeling / protein heterodimerization activity / chromatin binding / nucleolus / ATP hydrolysis activity / DNA binding / ATP binding / nucleus / plasma membrane
Similarity search - Function
HELLS, N-terminal / : / SNF2-like, N-terminal domain superfamily / SNF2, N-terminal / SNF2-related domain / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. ...HELLS, N-terminal / : / SNF2-like, N-terminal domain superfamily / SNF2, N-terminal / SNF2-related domain / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / Histone H3.3 / Histone H4 / Histone H2B / Probable histone H2A.7 / ATP-dependent DNA helicase DDM1
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
Xenopus laevis (African clawed frog)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsIpsaro, J.J. / Adams, D.W. / Joshua-Tor, L.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Cell / Year: 2023
Title: Chromatin remodeling of histone H3 variants by DDM1 underlies epigenetic inheritance of DNA methylation.
Authors: Seung Cho Lee / Dexter W Adams / Jonathan J Ipsaro / Jonathan Cahn / Jason Lynn / Hyun-Soo Kim / Benjamin Berube / Viktoria Major / Joseph P Calarco / Chantal LeBlanc / Sonali Bhattacharjee ...Authors: Seung Cho Lee / Dexter W Adams / Jonathan J Ipsaro / Jonathan Cahn / Jason Lynn / Hyun-Soo Kim / Benjamin Berube / Viktoria Major / Joseph P Calarco / Chantal LeBlanc / Sonali Bhattacharjee / Umamaheswari Ramu / Daniel Grimanelli / Yannick Jacob / Philipp Voigt / Leemor Joshua-Tor / Robert A Martienssen /
Abstract: Nucleosomes block access to DNA methyltransferase, unless they are remodeled by DECREASE in DNA METHYLATION 1 (DDM1), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 ...Nucleosomes block access to DNA methyltransferase, unless they are remodeled by DECREASE in DNA METHYLATION 1 (DDM1), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 promotes replacement of histone variant H3.3 by H3.1. In ddm1 mutants, DNA methylation is partly restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2 Å of DDM1 with a variant nucleosome reveals engagement with histone H3.3 near residues required for assembly and with the unmodified H4 tail. An N-terminal autoinhibitory domain inhibits activity, while a disulfide bond in the helicase domain supports activity. DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1, but is blocked by H4K16 acetylation. The male germline H3.3 variant MGH3/HTR10 is resistant to remodeling by DDM1 and acts as a placeholder nucleosome in sperm cells for epigenetic inheritance.
History
DepositionMay 5, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 9, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI ..._citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Sep 27, 2023Group: Database references / Refinement description / Category: citation / em_3d_fitting_list
Item: _citation.journal_volume / _citation.page_first / _em_3d_fitting_list.initial_refinement_model_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Probable histone H2A.7
B: Probable histone H2A.7
C: Histone H2B
D: Histone H2B
E: Histone H3.3
F: Histone H3.3
G: Histone H4
H: Histone H4
P: ATP-dependent DNA helicase DDM1
Y: DNA (sense strand)
Z: DNA (antisense strand)


Theoretical massNumber of molelcules
Total (without water)296,12111
Polymers296,12111
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 5 types, 9 molecules ABCDEFGHP

#1: Protein Probable histone H2A.7 / HTA6


Mass: 15997.942 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: At5g59870, MMN10.22, MMN10_90 / Plasmid: pET-3 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): pLysS / References: UniProt: Q9FJE8
#2: Protein Histone H2B /


Mass: 16474.459 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: At3g45980 / Plasmid: pET-3 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): pLysS / References: UniProt: Q0WT91
#3: Protein Histone H3.3 / H3F3A / Histone H3.2


Mass: 15440.144 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress)
Gene: HTR4, At4g40030, T5J17.200, HTR5, At4g40040, T5J17.210, HTR8, At5g10980, T30N20_250, T5K6.6
Plasmid: pET-3 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): pLysS / References: UniProt: P59169
#4: 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) / Plasmid: pET-3 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): pLysS / References: UniProt: P62799
#5: Protein ATP-dependent DNA helicase DDM1 / Protein CHROMATIN REMODELING 1 / AtCHR1 / CHR01 / Protein DECREASED DNA METHYLATION 1 / AtDDM1 / ...Protein CHROMATIN REMODELING 1 / AtCHR1 / CHR01 / Protein DECREASED DNA METHYLATION 1 / AtDDM1 / Protein SOMNIFEROUS 1 / SWI/SNF2-related matrix-associated actin-dependent regulator of chromatin DDM1


Mass: 86757.758 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: DDM1, CHA1, CHR1, SOM1, SOM4, At5g66750, MSN2.14 / Plasmid: pET-28 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): -RIPL / References: UniProt: Q9XFH4, DNA helicase

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DNA chain , 2 types, 2 molecules YZ

#6: DNA chain DNA (sense strand)


Mass: 45105.727 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#7: DNA chain DNA (antisense strand)


Mass: 45644.070 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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Arabidopsis DDM1 bound to nucleosome (H2A.W, H2B, H3.3, H4, with 147 bp DNA)COMPLEXComplex contains two copies of each histone (H2A.W, H2B, H3.3, and H4), 147 base pairs of DNA, and 1 copy of Arabidopsis DDM1all0MULTIPLE SOURCES
2DDM1COMPLEX#51RECOMBINANT
3Nucleosome core particleNucleosomeCOMPLEXComplex contains two copies of each histone (H2A.W, H2B, H3.3, and H4) and 147 base pairs of DNA#1-#4, #6-#71MULTIPLE SOURCES
4Histone octamerCOMPLEXContains two copies each of H2A.W, H2B, H3.3, and H4#1-#43RECOMBINANT
5DNA double helixNucleic acid double helixCOMPLEXGenerated by PCR amplification#6-#73RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.296 MDaNO
210.087 MDaNO
310.209 MDaNO
410.118 MDaNO
510.091 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDCellular location
12Arabidopsis thaliana (thale cress)3702nucleus
24Arabidopsis thaliana (thale cress)3702nucleus
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
12Escherichia coli (E. coli)562pLysSpET-28
24Escherichia coli (E. coli)562BL21(DE3)-RIPL
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
12 mMTrisC4H11NO31
210 mMHEPESC8H18N2O4S1
350 mMsodium chlorideNaClSodium chloride1
42 mMmagnesium chlorideMgCl21
51 mMATP-gamma-SC10H12N5O12P3S1
SpecimenConc.: 0.35 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: DDM1 and reconstituted nucleosomes were reconstituted in a 4:1 molar ratio. Crosslinking with 0.05% glutaraldehyde was performed for 15 minutes followed by quenching with 2 mM Tris, pH 8.0. ...Details: DDM1 and reconstituted nucleosomes were reconstituted in a 4:1 molar ratio. Crosslinking with 0.05% glutaraldehyde was performed for 15 minutes followed by quenching with 2 mM Tris, pH 8.0. ATP-gamma-S and MgCl2 were added at 1 and 2 mM, respectively.
Specimen supportGrid material: COPPER / Grid type: Quantifoil R0.6/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 K / Details: Blotted for 2.5 seconds before plunging.

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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 / Nominal magnification: 81000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4.8 sec. / Electron dose: 71.2 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8165
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 32

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Processing

EM software
IDNameVersionCategory
1Warp1.0.9particle selection
2EPU2.10.0.5image acquisition
4Warp1.0.9CTF correction
7Coot0.9.2-premodel fitting
9PHENIX1.19.2-4158-000model refinement
10cryoSPARC3.0.2+210831initial Euler assignment
11cryoSPARC3.0.2+210831final Euler assignment
12cryoSPARC3.0.2+210831classification
13cryoSPARC3.0.2+2108313D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 3788872
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 215066 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 57.8 / Space: REAL / Target criteria: Correlation coefficient
Atomic model building

3D fitting-ID: 1 / Accession code: 6UXW / Initial refinement model-ID: 1 / PDB-ID: 6UXW

6uxw

/ Source name: PDB / Type: experimental model

IDPdb chain-IDChain-ID
1AA
2BB
3CC
4DD
5EE
6FF
7GG
8HH
9II
10JJ
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00416378
ELECTRON MICROSCOPYf_angle_d0.75923299
ELECTRON MICROSCOPYf_dihedral_angle_d25.3566628
ELECTRON MICROSCOPYf_chiral_restr0.0452633
ELECTRON MICROSCOPYf_plane_restr0.0051961

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