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- EMDB-18793: Cryo-EM density map of DNMT3A1-DNMT3L on a human H2AKc119ub nucle... -

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Basic information

Entry
Database: EMDB / ID: EMD-18793
TitleCryo-EM density map of DNMT3A1-DNMT3L on a human H2AKc119ub nucleosome at 5.1 A resolution
Map dataDNMT3A1-DNMT3L_H2AKc119ub_unmaskedmap1
Sample
  • Complex: DNMT3A1-DNMT3L in complex with a human H2AKc119ub nucleosome wrapped with 195bp of Widom-601 positioning DNA
    • Complex: H2AKc119ub Nucleosome wrapped with 195 bp Widom601 DNA
      • Protein or peptide: Histone H3
      • Protein or peptide: Histone H2AK119C
      • Protein or peptide: ubiquitin
      • Protein or peptide: Histone H4
      • Protein or peptide: DNMT3L
      • Protein or peptide: Histone H2B
      • DNA: 195bp Widom601 DNA
    • Complex: DNMT3A1-DNMT3L complex
      • Protein or peptide: DNMT3A1
KeywordsChromatin / Nucleosome / methyltransferase / Ubiquitin / DNA BINDING PROTEIN
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.1 Å
AuthorsWapenaar H / Wilson MD
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust210493/Z/18/Z United Kingdom
CitationJournal: EMBO Rep / Year: 2024
Title: The N-terminal region of DNMT3A engages the nucleosome surface to aid chromatin recruitment.
Authors: Hannah Wapenaar / Gillian Clifford / Willow Rolls / Moira Pasquier / Hayden Burdett / Yujie Zhang / Gauri Deák / Juan Zou / Christos Spanos / Mark R D Taylor / Jacquie Mills / James A ...Authors: Hannah Wapenaar / Gillian Clifford / Willow Rolls / Moira Pasquier / Hayden Burdett / Yujie Zhang / Gauri Deák / Juan Zou / Christos Spanos / Mark R D Taylor / Jacquie Mills / James A Watson / Dhananjay Kumar / Richard Clark / Alakta Das / Devisree Valsakumar / Janice Bramham / Philipp Voigt / Duncan Sproul / Marcus D Wilson /
Abstract: DNA methyltransferase 3A (DNMT3A) plays a critical role in establishing and maintaining DNA methylation patterns in vertebrates. Here we structurally and biochemically explore the interaction of ...DNA methyltransferase 3A (DNMT3A) plays a critical role in establishing and maintaining DNA methylation patterns in vertebrates. Here we structurally and biochemically explore the interaction of DNMT3A1 with diverse modified nucleosomes indicative of different chromatin environments. A cryo-EM structure of the full-length DNMT3A1-DNMT3L complex with a H2AK119ub nucleosome reveals that the DNMT3A1 ubiquitin-dependent recruitment (UDR) motif interacts specifically with H2AK119ub and makes extensive contacts with the core nucleosome histone surface. This interaction facilitates robust DNMT3A1 binding to nucleosomes, and previously unexplained DNMT3A disease-associated mutations disrupt this interface. Furthermore, the UDR-nucleosome interaction synergises with other DNMT3A chromatin reading elements in the absence of histone ubiquitylation. H2AK119ub does not stimulate DNMT3A DNA methylation activity, as observed for the previously described H3K36me2 mark, which may explain low levels of DNA methylation on H2AK119ub marked facultative heterochromatin. This study highlights the importance of multivalent binding of DNMT3A to histone modifications and the nucleosome surface and increases our understanding of how DNMT3A1 chromatin recruitment occurs.
History
DepositionOct 31, 2023-
Header (metadata) releaseOct 23, 2024-
Map releaseOct 23, 2024-
UpdateDec 18, 2024-
Current statusDec 18, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18793.png

Downloads & links

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Map

FileDownload / File: emd_18793.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDNMT3A1-DNMT3L_H2AKc119ub_unmaskedmap1
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.66 Å/pix.
x 160 pix.
= 265.28 Å		1.66 Å/pix.
x 160 pix.
= 265.28 Å		1.66 Å/pix.
x 160 pix.
= 265.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.658 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00638
Minimum - Maximum-0.010626575 - 0.06812412
Average (Standard dev.)0.00031941046 (±0.0035905677)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 265.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18793_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Maskedmap1

Fileemd_18793_additional_1.map
AnnotationMaskedmap1
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap1 map1

Fileemd_18793_half_map_1.map
Annotationhalfmap1_map1
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap2 map1

Fileemd_18793_half_map_2.map
Annotationhalfmap2_map1
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : DNMT3A1-DNMT3L in complex with a human H2AKc119ub nucleosome wrap...

EntireName: DNMT3A1-DNMT3L in complex with a human H2AKc119ub nucleosome wrapped with 195bp of Widom-601 positioning DNA
Components
  • Complex: DNMT3A1-DNMT3L in complex with a human H2AKc119ub nucleosome wrapped with 195bp of Widom-601 positioning DNA
    • Complex: H2AKc119ub Nucleosome wrapped with 195 bp Widom601 DNA
      • Protein or peptide: Histone H3
      • Protein or peptide: Histone H2AK119C
      • Protein or peptide: ubiquitin
      • Protein or peptide: Histone H4
      • Protein or peptide: DNMT3L
      • Protein or peptide: Histone H2B
      • DNA: 195bp Widom601 DNA
    • Complex: DNMT3A1-DNMT3L complex
      • Protein or peptide: DNMT3A1

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Supramolecule #1: DNMT3A1-DNMT3L in complex with a human H2AKc119ub nucleosome wrap...

SupramoleculeName: DNMT3A1-DNMT3L in complex with a human H2AKc119ub nucleosome wrapped with 195bp of Widom-601 positioning DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 147 KDa

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Supramolecule #2: H2AKc119ub Nucleosome wrapped with 195 bp Widom601 DNA

SupramoleculeName: H2AKc119ub Nucleosome wrapped with 195 bp Widom601 DNA
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#4, #6-#7, #9
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: DNMT3A1-DNMT3L complex

SupramoleculeName: DNMT3A1-DNMT3L complex / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #8
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Histone H3

MacromoleculeName: Histone H3 / type: protein_or_peptide / ID: 1 / Details: C110A, C96S / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
ARTKQTARKS TGGKAPRKQL ATKAARKSAP ATGGVKKPHR YRPGTVALRE IRRYQKSTEL LIRKLPFQRL VREIAQDFKT DLRFQSSAVM ALQEASEAYL VGLFEDTNLA AIHAKRVTIM PKDIQLARRI RGERA

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Macromolecule #2: Histone H2AK119C

MacromoleculeName: Histone H2AK119C / type: protein_or_peptide / ID: 2
Details: K119C linked to ubiquitin via covalent acetone linker
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SGRGKQGGKA RAKAKTRSSR AGLQFPVGRV HRLLRKGNYA ERVGAGAPVY LAAVLEYLTA EILELAGNAA RDNKKTRIIP RHLQLAIRND EELNKLLGKV TIAQGGVLPN IQAVLLPKCT ESHHKAKGK

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Macromolecule #3: ubiquitin

MacromoleculeName: ubiquitin / type: protein_or_peptide / ID: 3
Details: G76C covalently liked with acetone linker to H2AK119C
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QIFVKTLTGK TITLEVEPSD TIENVKAKIQ DKEGIPPDQQ RLIFAGKQLE DGRTLSDYNI QKESTLHLVL RLRGC

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Macromolecule #4: Histone H4

MacromoleculeName: Histone H4 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SGRGKGGKGL GKGGAKRHRK VLRDNIQGIT KPAIRRLARR GGVKRISGLI YEETRGVLKV FLENVIRDAV TYTEHAKRKT VTAMDVVYAL KRQGRTLYGF GG

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Macromolecule #6: DNMT3L

MacromoleculeName: DNMT3L / type: protein_or_peptide / ID: 6 / Details: strep tag on C-terminus / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: SNMAAIPALD PEAEPSMDVI LVGSSELSSS VSPGTGRDLI AYEVKANQRN IEDICICCGS LQVHTQHPLF EGGICAPCKD KFLDALFLYD DDGYQSYCSI CCSGETLLIC GNPDCTRCYC FECVDSLVGP GTSGKVHAMS NWVCYLCLPS SRSGLLQRRR KWRSQLKAFY ...String:
SNMAAIPALD PEAEPSMDVI LVGSSELSSS VSPGTGRDLI AYEVKANQRN IEDICICCGS LQVHTQHPLF EGGICAPCKD KFLDALFLYD DDGYQSYCSI CCSGETLLIC GNPDCTRCYC FECVDSLVGP GTSGKVHAMS NWVCYLCLPS SRSGLLQRRR KWRSQLKAFY DRESENPLEM FETVPVWRRQ PVRVLSLFED IKKELTSLGF LESGSDPGQL KHVVDVTDTV RKDVEEWGPF DLVYGATPPL GHTCDRPPSW YLFQFHRLLQ YARPKPGSPR PFFWMFVDNL VLNKEDLDVA SRFLEMEPVT IPDVHGGSLQ NAVRVWSNIP AIRSRHWALV SEEELSLLAQ NKQSSKLAAK WPTKLVKNCF LPLREYFKYF STELTSSLGS SVWSHPQFEK

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Macromolecule #7: Histone H2B

MacromoleculeName: Histone H2B / type: protein_or_peptide / ID: 7 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
PEPAKSAPAP KKGSKKAVTK AQKKDGKKRK RSRKESYSVY VYKVLKQVHP DTGISSKAMG IMNSFVNDIF ERIAGEASRL AHYNKRSTIT SREIQTAVRL LLPGELAKHA VSEGTKAVTK YTSSK

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Macromolecule #8: DNMT3A1

MacromoleculeName: DNMT3A1 / type: protein_or_peptide / ID: 8 / Details: coexpressed with DNMT3L / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SNAMPAMPSS GPGDTSSSAA EREEDRKDGE EQEEPRGKEE RQEPSTTARK VGRPGRKRKH PPVESGDTPK DPAVISKSPS MAQDSGASEL LPNGDLEKRS EPQPEEGSPA GGQKGGAPAE GEGAAETLPE ASRAVENGCC TPKEGRGAPA EAGKEQKETN IESMKMEGSR ...String:
SNAMPAMPSS GPGDTSSSAA EREEDRKDGE EQEEPRGKEE RQEPSTTARK VGRPGRKRKH PPVESGDTPK DPAVISKSPS MAQDSGASEL LPNGDLEKRS EPQPEEGSPA GGQKGGAPAE GEGAAETLPE ASRAVENGCC TPKEGRGAPA EAGKEQKETN IESMKMEGSR GRLRGGLGWE SSLRQRPMPR LTFQAGDPYY ISKRKRDEWL ARWKREAEKK AKVIAGMNAV EENQGPGESQ KVEEASPPAV QQPTDPASPT VATTPEPVGS DAGDKNATKA GDDEPEYEDG RGFGIGELVW GKLRGFSWWP GRIVSWWMTG RSRAAEGTRW VMWFGDGKFS VVCVEKLMPL SSFCSAFHQA TYNKQPMYRK AIYEVLQVAS SRAGKLFPVC HDSDESDTAK AVEVQNKPMI EWALGGFQPS GPKGLEPPEE EKNPYKEVYT DMWVEPEAAA YAPPPPAKKP RKSTAEKPKV KEIIDERTRE RLVYEVRQKC RNIEDICISC GSLNVTLEHP LFVGGMCQNC KNCFLECAYQ YDDDGYQSYC TICCGGREVL MCGNNNCCRC FCVECVDLLV GPGAAQAAIK EDPWNCYMCG HKGTYGLLRR REDWPSRLQM FFANNHDQEF DPPKVYPPVP AEKRKPIRVL SLFDGIATGL LVLKDLGIQV DRYIASEVCE DSITVGMVRH QGKIMYVGDV RSVTQKHIQE WGPFDLVIGG SPCNDLSIVN PARKGLYEGT GRLFFEFYRL LHDARPKEGD DRPFFWLFEN VVAMGVSDKR DISRFLESNP VMIDAKEVSA AHRARYFWGN LPGMNRPLAS TVNDKLELQE CLEHGRIAKF SKVRTITTRS NSIKQGKDQH FPVFMNEKED ILWCTEMERV FGFPVHYTDV SNMSRLARQR LLGRSWSVPV IRHLFAPLKE YFACV

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Macromolecule #9: 195bp Widom601 DNA

MacromoleculeName: 195bp Widom601 DNA / type: dna / ID: 9 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString: TGGAGAATCC CGGTGCCGAG GCCGCTCAAT TGGTCGTAGA CAGCTCTAGC ACCGCTTAAA CGCACGTACG CGCTGTCCCC CGCGTTTTAA CCGCCAAGGG GATTACTCCC TAGTCTCCAG GCACGTGTCA GATATATACA TCCTGTCACC ATACGCCCTA ATTAGAGGCG ...String:
TGGAGAATCC CGGTGCCGAG GCCGCTCAAT TGGTCGTAGA CAGCTCTAGC ACCGCTTAAA CGCACGTACG CGCTGTCCCC CGCGTTTTAA CCGCCAAGGG GATTACTCCC TAGTCTCCAG GCACGTGTCA GATATATACA TCCTGTCACC ATACGCCCTA ATTAGAGGCG TAATCCCCCA GTTCGCGCGC CCACC

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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

Concentration0.13 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
15.0 mMC8H18N2O4SHEPES
65.0 mMNaClsodium chloride
1.0 mMC4H10O2S2Dithiothreitol
0.1 mMC15H22N6O5SS-Adenosyl methionine

Details: 15 mM HEPES pH 7.5, 65 mM NaCl, 1 mM DTT, 0.1 mM S-Adenosyl methionine
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.038 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 6969 / Average electron dose: 61.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 105000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsMovies were motion corrected with MotionCor2
Particle selectionNumber selected: 1824711
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4) / Number images used: 203347
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4)
Final 3D classificationNumber classes: 1 / Avg.num./class: 203347 / Software - Name: RELION (ver. 4)
FSC plot (resolution estimation)

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