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- PDB-7xib: Cryo-EM structure of human DNMT1 (aa:351-1616) in complex with ub... -

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Basic information

Entry
Database: PDB / ID: 7xib
TitleCryo-EM structure of human DNMT1 (aa:351-1616) in complex with ubiquitinated H3 and hemimethylated DNA analog (CXXC-disordered form)
Components
  • DNA (5'-D(*AP*CP*TP*TP*AP*(5CM)P*GP*GP*AP*AP*GP*G)-3')
  • DNA (5'-D(*CP*CP*TP*TP*CP*(C55)P*GP*TP*AP*AP*GP*T)-3')
  • DNA (cytosine-5)-methyltransferase 1
KeywordsTRANSFERASE/DNA / DNA methyltransferase / TRANSFERASE-DNA COMPLEX
Function / homology
Function and homology information


negative regulation of vascular associated smooth muscle cell differentiation involved in phenotypic switching / : / epigenetic programming of gene expression / cellular response to bisphenol A / negative regulation of vascular associated smooth muscle cell apoptotic process / DNA-methyltransferase activity / DNA (cytosine-5-)-methyltransferase / DNA (cytosine-5-)-methyltransferase activity / DNA methylation-dependent heterochromatin formation / SUMOylation of DNA methylation proteins ...negative regulation of vascular associated smooth muscle cell differentiation involved in phenotypic switching / : / epigenetic programming of gene expression / cellular response to bisphenol A / negative regulation of vascular associated smooth muscle cell apoptotic process / DNA-methyltransferase activity / DNA (cytosine-5-)-methyltransferase / DNA (cytosine-5-)-methyltransferase activity / DNA methylation-dependent heterochromatin formation / SUMOylation of DNA methylation proteins / negative regulation of gene expression via chromosomal CpG island methylation / female germ cell nucleus / methyl-CpG binding / pericentric heterochromatin / positive regulation of vascular associated smooth muscle cell proliferation / DNA methylation / replication fork / PRC2 methylates histones and DNA / Defective pyroptosis / promoter-specific chromatin binding / cellular response to amino acid stimulus / NoRC negatively regulates rRNA expression / negative regulation of gene expression / DNA-templated transcription / positive regulation of gene expression / negative regulation of transcription by RNA polymerase II / DNA binding / RNA binding / zinc ion binding / nucleoplasm / nucleus
Similarity search - Function
DNA (cytosine-5)-methyltransferase 1-like / DNA (cytosine-5)-methyltransferase 1, replication foci domain / Cytosine specific DNA methyltransferase replication foci domain / DMAP1-binding Domain / DMAP1-binding Domain / DMAP1-binding domain / DMAP1-binding domain profile. / DNA methylase, C-5 cytosine-specific, conserved site / C-5 cytosine-specific DNA methylases C-terminal signature. / DNA methylase, C-5 cytosine-specific, active site ...DNA (cytosine-5)-methyltransferase 1-like / DNA (cytosine-5)-methyltransferase 1, replication foci domain / Cytosine specific DNA methyltransferase replication foci domain / DMAP1-binding Domain / DMAP1-binding Domain / DMAP1-binding domain / DMAP1-binding domain profile. / DNA methylase, C-5 cytosine-specific, conserved site / C-5 cytosine-specific DNA methylases C-terminal signature. / DNA methylase, C-5 cytosine-specific, active site / C-5 cytosine-specific DNA methylases active site. / CXXC zinc finger domain / Zinc finger, CXXC-type / Zinc finger CXXC-type profile. / C-5 cytosine-specific DNA methylase (Dnmt) domain profile. / C-5 cytosine methyltransferase / C-5 cytosine-specific DNA methylase / Bromo adjacent homology (BAH) domain superfamily / Bromo adjacent homology domain / Bromo adjacent homology (BAH) domain / BAH domain / BAH domain profile. / S-adenosyl-L-methionine-dependent methyltransferase superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (cytosine-5)-methyltransferase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.23 Å
AuthorsOnoda, H. / Kikuchi, A. / Kori, S. / Yoshimi, S. / Yamagata, A. / Arita, K.
Funding support Japan, 4items
OrganizationGrant numberCountry
Ministry of Education, Culture, Sports, Science and Technology (Japan)JP18H02392 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)JP19H05294 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)JP19H05741 Japan
Japan Society for the Promotion of Science (JSPS)SK201904 Japan
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis for activation of DNMT1.
Authors: Amika Kikuchi / Hiroki Onoda / Kosuke Yamaguchi / Satomi Kori / Shun Matsuzawa / Yoshie Chiba / Shota Tanimoto / Sae Yoshimi / Hiroki Sato / Atsushi Yamagata / Mikako Shirouzu / Naruhiko ...Authors: Amika Kikuchi / Hiroki Onoda / Kosuke Yamaguchi / Satomi Kori / Shun Matsuzawa / Yoshie Chiba / Shota Tanimoto / Sae Yoshimi / Hiroki Sato / Atsushi Yamagata / Mikako Shirouzu / Naruhiko Adachi / Jafar Sharif / Haruhiko Koseki / Atsuya Nishiyama / Makoto Nakanishi / Pierre-Antoine Defossez / Kyohei Arita /
Abstract: DNMT1 is an essential enzyme that maintains genomic DNA methylation, and its function is regulated by mechanisms that are not yet fully understood. Here, we report the cryo-EM structure of human ...DNMT1 is an essential enzyme that maintains genomic DNA methylation, and its function is regulated by mechanisms that are not yet fully understood. Here, we report the cryo-EM structure of human DNMT1 bound to its two natural activators: hemimethylated DNA and ubiquitinated histone H3. We find that a hitherto unstudied linker, between the RFTS and CXXC domains, plays a key role for activation. It contains a conserved α-helix which engages a crucial "Toggle" pocket, displacing a previously described inhibitory linker, and allowing the DNA Recognition Helix to spring into the active conformation. This is accompanied by large-scale reorganization of the inhibitory RFTS and CXXC domains, allowing the enzyme to gain full activity. Our results therefore provide a mechanistic basis for the activation of DNMT1, with consequences for basic research and drug design.
History
DepositionApr 12, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 30, 2022Provider: repository / Type: Initial release
Revision 1.1Dec 7, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DNA (cytosine-5)-methyltransferase 1
B: DNA (5'-D(*AP*CP*TP*TP*AP*(5CM)P*GP*GP*AP*AP*GP*G)-3')
C: DNA (5'-D(*CP*CP*TP*TP*CP*(C55)P*GP*TP*AP*AP*GP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)150,6425
Polymers150,5113
Non-polymers1312
Water3,531196
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, SDS-PAGE and Agarose Gel Electrophoresis of the gel filtration fraction indicate that DNMT1, H3Ub2 and hemi-methyl DNA analog form ternary complex.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area5830 Å2
ΔGint-27 kcal/mol
Surface area36930 Å2

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Components

#1: Protein DNA (cytosine-5)-methyltransferase 1 / Dnmt1


Mass: 143106.000 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DNMT1 / Plasmid: pFastBac / Cell line (production host): sf-9 / Production host: Baculovirus expression vector pFastBac1-HM
References: UniProt: P26358, DNA (cytosine-5-)-methyltransferase
#2: DNA chain DNA (5'-D(*AP*CP*TP*TP*AP*(5CM)P*GP*GP*AP*AP*GP*G)-3')


Mass: 3725.469 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(*CP*CP*TP*TP*CP*(C55)P*GP*TP*AP*AP*GP*T)-3')


Mass: 3679.464 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 196 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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
IDNameTypeEntity IDParent-IDSource
1DNMT1:H3Ub2:DNAmCG/fCG Ternary complex of DNMT1 with hemi methylated DNA analog and H3Ub2COMPLEX#1-#30MULTIPLE SOURCES
2DNA (cytosine-5)-methyltransferase 1COMPLEX#11RECOMBINANT
3DNACOMPLEX#2-#31SYNTHETIC
Molecular weightValue: 0.17 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Baculovirus expression vector pFastBac1-HM
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM2-Amino-2-hydroxymethyl-propane-1,3-diolTris-HClTris1
2250 mMSodium chlorideNaClSodium chloride1
35 mMDithiothreitolDTT1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: This sample was monodisperse by Size-exclusion chromatography
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Blot for 4 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 49 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4068
Image scansWidth: 4092 / Height: 5760

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.17.1_3660refinement
PHENIX1.17.1_3660refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARC3.2.0particle selectionBlob picker was used to automatically select particle images.
4cryoSPARC3.2.0CTF correctionPatch CTF
7PHENIX1.17.1model fitting
9cryoSPARC3.2.0initial Euler assignmentAb-initio
10cryoSPARC3.2.0final Euler assignmentNon-uniform Refinement
11cryoSPARC3.2.0classification3D Variability
12cryoSPARC3.2.03D reconstructionNon-uniform Refinement
13PHENIX1.17.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3798046
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 897446 / Algorithm: EXACT BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
16X9I

6x9i

A1730-1600
26X9I

6x9i

B11-12
36X9I

6x9i

C113-24
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 40.28 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00447200
ELECTRON MICROSCOPYf_angle_d0.69199847
ELECTRON MICROSCOPYf_chiral_restr0.04791048
ELECTRON MICROSCOPYf_plane_restr0.00491201
ELECTRON MICROSCOPYf_dihedral_angle_d24.30981135

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