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- EMDB-33200: Cryo-EM structure of human DNMT1 (aa:351-1616) in complex with ub... -

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Basic information

Entry
Database: EMDB / ID: EMD-33200
TitleCryo-EM structure of human DNMT1 (aa:351-1616) in complex with ubiquitinated H3 and hemimethylated DNA analog (CXXC-ordered form)
Map data
Sample
  • Complex: DNMT1:H3Ub2:DNAmCG/fCGTernary complex of DNMT1 with hemi methylated DNA analog and H3Ub2
    • Complex: DNA (cytosine-5)-methyltransferase 1
      • Protein or peptide: DNA (cytosine-5)-methyltransferase 1
    • Complex: DNA
      • DNA: DNA (5'-D(*AP*CP*TP*TP*AP*(5CM)P*GP*GP*AP*AP*GP*G)-3')
      • DNA: DNA (5'-D(*CP*CP*TP*TP*CP*(C55)P*GP*TP*AP*AP*GP*T)-3')
  • Ligand: ZINC ION
  • Ligand: S-ADENOSYL-L-HOMOCYSTEINE
  • Ligand: water
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 (cytosine-5)-methyltransferase 1
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.52 Å
AuthorsOnoda H / Kikuchi A / Kori S / Yoshimi S / Yamagata A / Arita K
Funding support Japan, 4 items
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, 2022-
Header (metadata) releaseNov 30, 2022-
Map releaseNov 30, 2022-
UpdateDec 7, 2022-
Current statusDec 7, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33200.png

Downloads & links

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Map

FileDownload / File: emd_33200.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.4836217 - 2.9105258
Average (Standard dev.)-0.00019711751 (±0.062899604)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 212.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_33200_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Additional map: #1

Fileemd_33200_additional_1.map
Projections & Slices
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Half map: #2

Fileemd_33200_half_map_1.map
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Half map: #1

Fileemd_33200_half_map_2.map
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Sample components

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Entire : DNMT1:H3Ub2:DNAmCG/fCGTernary complex of DNMT1 with hemi methylat...

EntireName: DNMT1:H3Ub2:DNAmCG/fCGTernary complex of DNMT1 with hemi methylated DNA analog and H3Ub2
Components
  • Complex: DNMT1:H3Ub2:DNAmCG/fCGTernary complex of DNMT1 with hemi methylated DNA analog and H3Ub2
    • Complex: DNA (cytosine-5)-methyltransferase 1
      • Protein or peptide: DNA (cytosine-5)-methyltransferase 1
    • Complex: DNA
      • DNA: DNA (5'-D(*AP*CP*TP*TP*AP*(5CM)P*GP*GP*AP*AP*GP*G)-3')
      • DNA: DNA (5'-D(*CP*CP*TP*TP*CP*(C55)P*GP*TP*AP*AP*GP*T)-3')
  • Ligand: ZINC ION
  • Ligand: S-ADENOSYL-L-HOMOCYSTEINE
  • Ligand: water

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Supramolecule #1: DNMT1:H3Ub2:DNAmCG/fCGTernary complex of DNMT1 with hemi methylat...

SupramoleculeName: DNMT1:H3Ub2:DNAmCG/fCGTernary complex of DNMT1 with hemi methylated DNA analog and H3Ub2
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 170 KDa

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Supramolecule #2: DNA (cytosine-5)-methyltransferase 1

SupramoleculeName: DNA (cytosine-5)-methyltransferase 1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3

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Macromolecule #1: DNA (cytosine-5)-methyltransferase 1

MacromoleculeName: DNA (cytosine-5)-methyltransferase 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA (cytosine-5-)-methyltransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 143.106 KDa
Recombinant expressionOrganism: Baculovirus expression vector pFastBac1-HM
SequenceString: PKCIQCGQYL DDPDLKYGQH PPDAVDEPQM LTNEKLSIFD ANESGFESYE ALPQHKLTCF SVYCKHGHLC PIDTGLIEKN IELFFSGSA KPIYDDDPSL EGGVNGKNLG PINEWWITGF DGGEKALIGF STSFAEYILM DPSPEYAPIF GLMQEKIYIS K IVVEFLQS ...String:
PKCIQCGQYL DDPDLKYGQH PPDAVDEPQM LTNEKLSIFD ANESGFESYE ALPQHKLTCF SVYCKHGHLC PIDTGLIEKN IELFFSGSA KPIYDDDPSL EGGVNGKNLG PINEWWITGF DGGEKALIGF STSFAEYILM DPSPEYAPIF GLMQEKIYIS K IVVEFLQS NSDSTYEDLI NKIETTVPPS GLNLNRFTED SLLRHAQFVV EQVESYDEAG DSDEQPIFLT PCMRDLIKLA GV TLGQRRA QARRQTIRHS TREKDRGPTK ATTTKLVYQI FDTFFAEQIE KDDREDKENA FKRRRCGVCE VCQQPECGKC KAC KDMVKF GGSGRSKQAC QERRCPNMAM KEADDDEEVD DNIPEMPSPK KMHQGKKKKQ NKNRISWVGE AVKTDGKKSY YKKV CIDAE TLEVGDCVSV IPDDSSKPLY LARVTALWED SSNGQMFHAH WFCAGTDTVL GATSDPLELF LVDECEDMQL SYIHS KVKV IYKAPSENWA MEGGMDPESL LEGDDGKTYF YQLWYDQDYA RFESPPKTQP TEDNKFKFCV SCARLAEMRQ KEIPRV LEQ LEDLDSRVLY YSATKNGILY RVGDGVYLPP EAFTFNIKLS SPVKRPRKEP VDEDLYPEHY RKYSDYIKGS NLDAPEP YR IGRIKEIFCP KKSNGRPNET DIKIRVNKFY RPENTHKSTP ASYHADINLL YWSDEEAVVD FKAVQGRCTV EYGEDLPE C VQVYSMGGPN RFYFLEAYNA KSKSFEDPPN HARSPGNKGK GKGKGKGKPK SQACEPSEPE IEIKLPKLRT LDVFSGCGG LSEGFHQAGI SDTLWAIEMW DPAAQAFRLN NPGSTVFTED CNILLKLVMA GETTNSRGQR LPQKGDVEML CGGPPCQGFS GMNRFNSRT YSKFKNSLVV SFLSYCDYYR PRFFLLENVR NFVSFKRSMV LKLTLRCLVR MGYQCTFGVL QAGQYGVAQT R RRAIILAA APGEKLPLFP EPLHVFAPRA CQLSVVVDDK KFVSNITRLS SGPFRTITVR DTMSDLPEVR NGASALEISY NG EPQSWFQ RQLRGAQYQP ILRDHICKDM SALVAARMRH IPLAPGSDWR DLPNIEVRLS DGTMARKLRY THHDRKNGRS SSG ALRGVC SCVEAGKACD PAARQFNTLI PWCLPHTGNR HNHWAGLYGR LEWDGFFSTT VTNPEPMGKQ GRVLHPEQHR VVSV RECAR SQGFPDTYRL FGNILDKHRQ VGNAVPPPLA KAIGLEIKLC MLAKARESAS AKIKEEEAAK D

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Macromolecule #2: DNA (5'-D(*AP*CP*TP*TP*AP*(5CM)P*GP*GP*AP*AP*GP*G)-3')

MacromoleculeName: DNA (5'-D(*AP*CP*TP*TP*AP*(5CM)P*GP*GP*AP*AP*GP*G)-3')
type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.725469 KDa
SequenceString:
(DA)(DC)(DT)(DT)(DA)(5CM)(DG)(DG)(DA)(DA) (DG)(DG)

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Macromolecule #3: DNA (5'-D(*CP*CP*TP*TP*CP*(C55)P*GP*TP*AP*AP*GP*T)-3')

MacromoleculeName: DNA (5'-D(*CP*CP*TP*TP*CP*(C55)P*GP*TP*AP*AP*GP*T)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.679464 KDa
SequenceString:
(DC)(DC)(DT)(DT)(DC)(EIX)(DG)(DT)(DA)(DA) (DG)(DT)

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #5: S-ADENOSYL-L-HOMOCYSTEINE

MacromoleculeName: S-ADENOSYL-L-HOMOCYSTEINE / type: ligand / ID: 5 / Number of copies: 1 / Formula: SAH
Molecular weightTheoretical: 384.411 Da
Chemical component information

ChemComp-SAH:
S-ADENOSYL-L-HOMOCYSTEINE / S-Adenosyl-L-homocysteine

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 232 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

Concentration0.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMTris-HClTris2-Amino-2-hydroxymethyl-propane-1,3-diol
250.0 mMNaClSodium chlorideSodium chloride
5.0 mMDTTDithiothreitol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 4 seconds before plunging.
DetailsThis sample was monodisperse by Size-exclusion chromatography

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 4092 pixel / Digitization - Dimensions - Height: 5760 pixel / Number grids imaged: 1 / Number real images: 4068 / Average electron dose: 49.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3798046
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0) / Software - details: Ab-initio
Final 3D classificationNumber classes: 5 / Avg.num./class: 182833 / Software - Name: cryoSPARC (ver. 3.2.0) / Software - details: 3D Variability
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0) / Software - details: Non-uniform Refinement
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: EXACT BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.52 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Software - details: Non-uniform Refinement / Number images used: 138662
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6x9i

chain_id: A, residue_range: 730-1600

6x9i

chain_id: B, residue_range: 1-12

6x9i

chain_id: C, residue_range: 13-24
RefinementSpace: REAL / Protocol: BACKBONE TRACE
Output model

PDB-7xi9:
Cryo-EM structure of human DNMT1 (aa:351-1616) in complex with ubiquitinated H3 and hemimethylated DNA analog (CXXC-ordered form)

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