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- EMDB-33298: Cryo-EM map of apo-DNMT1 (aa:351-1616) -

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Basic information

Entry
Database: EMDB / ID: EMD-33298
TitleCryo-EM map of apo-DNMT1 (aa:351-1616)
Map data
Sample
  • Complex: human DNMT1, 351_1616 with H3Ub2
    • Complex: DNA (cytosine-5)-methyltransferase 1
      • Protein or peptide: DNA (cytosine-5)-methyltransferase 1
    • Complex: Histone H3.1Histone H3
      • Protein or peptide: Histone H3.1 K14R/K27R/K36R/K37W
    • Complex: H3Ub2
      • Protein or peptide: Ubiquitin
KeywordsDNA methyltransferase / TRANSFERASE
Function / homology:
Function and homology information
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.52 Å
AuthorsOnoda H / Kikuchi A / Kori S / Yoshimi S / Yamagata A / Matsuzawa S / 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 26, 2022-
Header (metadata) releaseNov 30, 2022-
Map releaseNov 30, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33298.png

Downloads & links

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Map

FileDownload / File: emd_33298.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.477 Å
Density
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-0.8638912 - 2.0974483
Average (Standard dev.)-0.00043712437 (±0.033189967)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 378.112 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_33298_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_33298_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_33298_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : human DNMT1, 351_1616 with H3Ub2

EntireName: human DNMT1, 351_1616 with H3Ub2
Components
  • Complex: human DNMT1, 351_1616 with H3Ub2
    • Complex: DNA (cytosine-5)-methyltransferase 1
      • Protein or peptide: DNA (cytosine-5)-methyltransferase 1
    • Complex: Histone H3.1Histone H3
      • Protein or peptide: Histone H3.1 K14R/K27R/K36R/K37W
    • Complex: H3Ub2
      • Protein or peptide: Ubiquitin

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Supramolecule #1: human DNMT1, 351_1616 with H3Ub2

SupramoleculeName: human DNMT1, 351_1616 with H3Ub2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 160 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
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Histone H3.1

SupramoleculeName: Histone H3.1 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: H3Ub2

SupramoleculeName: H3Ub2 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3

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

MacromoleculeName: DNA (cytosine-5)-methyltransferase 1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: DNA (cytosine-5-)-methyltransferase
Source (natural)Organism: Homo sapiens (human) / Strain: Isoform 1
Recombinant expressionOrganism: Baculovirus expression vector pFastBac1-HM
SequenceString: PKCIQCGQYL DDPDLKYGQH PPDAVDEPQ MLTNEKLSIF D ANESGFES YEALPQHKLT CF SVYCKHG HLCPIDTGLI EKN IELFFS GSAKPIYDDD PSLE GGVNG KNLGPINEWW ITGFD GGEK ALIGFSTSFA EYILMD PSP EYAPIFGLMQ EKIYISK IV ...String:
PKCIQCGQYL DDPDLKYGQH PPDAVDEPQ MLTNEKLSIF D ANESGFES YEALPQHKLT CF SVYCKHG HLCPIDTGLI EKN IELFFS GSAKPIYDDD PSLE GGVNG KNLGPINEWW ITGFD GGEK ALIGFSTSFA EYILMD PSP EYAPIFGLMQ EKIYISK IV VEFLQSNSDS TYEDLINK I ETTVPPSGLN LNRFTEDSL LRHAQFVVEQ VESYDEAGDS DEQPIFLTP CMRDLIKLAG V TLGQRRAQ ARRQTIRHST RE KDRGPTK ATTTKLVYQI FDT FFAEQI EKDDREDKEN AFKR RRCGV CEVCQQPECG KCKAC KDMV KFGGSGRSKQ ACQERR CPN MAMKEADDDE EVDDNIP EM PSPKKMHQGK KKKQNKNR I SWVGEAVKTD GKKSYYKKV CIDAETLEVG DCVSVIPDDS SKPLYLARV TALWEDSSNG Q MFHAHWFC AGTDTVLGAT SD PLELFLV DECEDMQLSY IHS KVKVIY KAPSENWAME GGMD PESLL EGDDGKTYFY QLWYD QDYA RFESPPKTQP TEDNKF KFC VSCARLAEMR QKEIPRV LE QLEDLDSRVL YYSATKNG I LYRVGDGVYL PPEAFTFNI KLSSPVKRPR KEPVDEDLYP EHYRKYSDY IKGSNLDAPE P YRIGRIKE IFCPKKSNGR PN ETDIKIR VNKFYRPENT HKS TPASYH ADINLLYWSD EEAV VDFKA VQGRCTVEYG EDLPE CVQV YSMGGPNRFY FLEAYN AKS KSFEDPPNHA RSPGNKG KG KGKGKGKPKS QACEPSEP E IEIKLPKLRT LDVFSGCGG LSEGFHQAGI SDTLWAIEMW DPAAQAFRL NNPGSTVFTE D CNILLKLV MAGETTNSRG QR LPQKGDV EMLCGGPPCQ GFS GMNRFN SRTYSKFKNS LVVS FLSYC DYYRPRFFLL ENVRN FVSF KRSMVLKLTL RCLVRM GYQ CTFGVLQAGQ YGVAQTR RR AIILAAAPGE KLPLFPEP L HVFAPRACQL SVVVDDKKF VSNITRLSSG PFRTITVRDT MSDLPEVRN GASALEISYN G EPQSWFQR QLRGAQYQPI LR DHICKDM SALVAARMRH IPL APGSDW RDLPNIEVRL SDGT MARKL RYTHHDRKNG RSSSG ALRG VCSCVEAGKA CDPAAR QFN TLIPWCLPHT GNRHNHW AG LYGRLEWDGF FSTTVTNP E PMGKQGRVLH PEQHRVVSV RECARSQGFP DTYRLFGNIL DKHRQVGNA VPPPLAKAIG L EIKLCMLA KARESASAKI KE EEAAKD

UniProtKB: UNIPROTKB: NP_001370.1

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Macromolecule #2: Histone H3.1 K14R/K27R/K36R/K37W

MacromoleculeName: Histone H3.1 K14R/K27R/K36R/K37W / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
ARTKQTARK STGGRAPRKQ LATKAARRS A PATGGVRW

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Macromolecule #3: Ubiquitin

MacromoleculeName: Ubiquitin / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString:
MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHL VLRLRGG

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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

BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMTris-HClTris2-Amino-2-hydroxymethyl-propane-1,3-diol
250.0 mMNaClSodium chlorideSodium chloride
0.5 mMDTTDithiothreitol
0.01 mMZn(OAc)2Zinc acetate
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 TECNAI ARCTICA
Electron beamAcceleration voltage: 200 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: 1.4000000000000001 µm / Nominal defocus min: 0.8 µm
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Number grids imaged: 1 / Number real images: 1869 / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2463410
Details: Blob picker using circular blob of 80pix, 105pix 130pix diameter. the particle was filtered by Local power larger than 397.000.
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1) / Software - details: Ab-initio
Final 3D classificationNumber classes: 5 / Avg.num./class: 147047 / Software - Name: cryoSPARC (ver. 3.3.1) / Software - details: 3D variability
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1) / Software - details: Non-uniform Refinement
Final reconstructionNumber classes used: 4 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: EXACT BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.52 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Software - details: Non-uniform Refinement / Number images used: 735233
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4wxx


Chain - Chain ID: B / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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