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- EMDB-50802: Human DNA methyltransferase 1 productive DNA complex in presence ... -

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Basic information

Entry
Database: EMDB / ID: EMD-50802
TitleHuman DNA methyltransferase 1 productive DNA complex in presence of H3Ub2-peptide
Map dataHsDNMT1 in complex with 5FDNA in presence of H3Ub2-peptide single particle cryoEM map
Sample
  • Complex: Human DNA methyltransferase 1 bound to 5FDNA and in presence of H3Ub2-peptide
KeywordsDNA methylation / methyltransferase / TRANSFERASE
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsDe I / Weidenhausen J / Mueller CW / Concha N
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: PLoS One / Year: 2024
Title: Structural insight into the DNMT1 reaction cycle by cryo-electron microscopy.
Authors: Inessa De / Jonas Weidenhausen / Nestor Concha / Christoph W Müller /
Abstract: DNMT1 is an essential DNA methyltransferase that catalyzes the transfer of methyl groups to CpG islands in DNA and generates a prominent epigenetic mark. The catalytic activity of DNMT1 relies on its ...DNMT1 is an essential DNA methyltransferase that catalyzes the transfer of methyl groups to CpG islands in DNA and generates a prominent epigenetic mark. The catalytic activity of DNMT1 relies on its conformational plasticity and ability to change conformation from an auto-inhibited to an activated state. Here, we present four cryo-EM reconstructions of apo DNMT1 and DNTM1: non-productive DNA, DNTM1: H3Ub2-peptide, DNTM1: productive DNA complexes. Our structures demonstrate the flexibility of DNMT1's N-terminal regulatory domains during the transition from an apo 'auto-inhibited' to a DNA-bound 'non-productive' and finally a DNA-bound 'productive' state of DNMT1. Furthermore, we address the regulation of DNMT1's methyltransferase activity by a DNMT1-selective small-molecule inhibitor and ubiquitinated histone H3. We observe that DNMT1 binds DNA in a 'non-productive' state despite the presence of the inhibitor and present the cryo-EM reconstruction of full-length DNMT1 in complex with a di-ubiquitinated H3 peptide analogue. Taken together, our results provide structural insights into the reaction cycle of DNMT1.
History
DepositionJun 26, 2024-
Header (metadata) releaseJul 24, 2024-
Map releaseJul 24, 2024-
UpdateSep 11, 2024-
Current statusSep 11, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50802.png

Downloads & links

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Map

FileDownload / File: emd_50802.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHsDNMT1 in complex with 5FDNA in presence of H3Ub2-peptide single particle cryoEM map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.88 Å/pix.
x 128 pix.
= 240.64 Å		1.88 Å/pix.
x 128 pix.
= 240.64 Å		1.88 Å/pix.
x 128 pix.
= 240.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel size
XYZ
EMDB info.1.0411.0411.041
CCP4 map header1.881.881.88
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.17391919 - 0.6819722
Average (Standard dev.)0.0010921516 (±0.020592207)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 291.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half-map A

Fileemd_50802_half_map_1.map
Annotationhalf-map A
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 DNA methyltransferase 1 bound to 5FDNA and in presence of H...

EntireName: Human DNA methyltransferase 1 bound to 5FDNA and in presence of H3Ub2-peptide
Components
  • Complex: Human DNA methyltransferase 1 bound to 5FDNA and in presence of H3Ub2-peptide

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Supramolecule #1: Human DNA methyltransferase 1 bound to 5FDNA and in presence of H...

SupramoleculeName: Human DNA methyltransferase 1 bound to 5FDNA and in presence of H3Ub2-peptide
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)

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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

Concentration0.43 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 200 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 45 sec.
VitrificationCryogen name: ETHANE
Details0.43 mg/mL DNMT1 was incubated with 5-fold molar excess of a histone 3 di-ubiquitinated peptide analogue, followed by 8-fold molar excess of 5FDNA and 50-fold molar excess SAM. The complex was purified by ion exchange chromatography

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 130000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 133447
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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