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- EMDB-33836: CryoEM structure of Arabidopsis ROS1 in complex with a covalent-l... -

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Basic information

Entry
Database: EMDB / ID: EMD-33836
TitleCryoEM structure of Arabidopsis ROS1 in complex with a covalent-linked reaction intermediate at 3.9 Angstroms resolution
Map data
Sample
  • Complex: covalent-linked reaction intermediate of Arabidopsis ROS1 with dsDNA
    • Complex: ROS1
      • Protein or peptide: Sex-determining region Y protein,DNA glycosylase/AP lyase ROS1
    • Complex: dsDNA
      • DNA: DNA (40-MER)
      • DNA: DNA (40-MER)
  • Ligand: IRON/SULFUR CLUSTER
KeywordsROS1 / DNA glycosylase / DNA demethylation / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


DNA demethylase activity / chromosomal 5-methylcytosine DNA demethylation pathway / positive regulation of male gonad development / Transcriptional regulation of testis differentiation / sex differentiation / DNA N-glycosylase activity / male sex determination / defense response to fungus / DNA-(apurinic or apyrimidinic site) endonuclease activity / class I DNA-(apurinic or apyrimidinic site) endonuclease activity ...DNA demethylase activity / chromosomal 5-methylcytosine DNA demethylation pathway / positive regulation of male gonad development / Transcriptional regulation of testis differentiation / sex differentiation / DNA N-glycosylase activity / male sex determination / defense response to fungus / DNA-(apurinic or apyrimidinic site) endonuclease activity / class I DNA-(apurinic or apyrimidinic site) endonuclease activity / DNA-(apurinic or apyrimidinic site) lyase / Deactivation of the beta-catenin transactivating complex / base-excision repair / brain development / neuron differentiation / 4 iron, 4 sulfur cluster binding / DNA-binding transcription activator activity, RNA polymerase II-specific / DNA-binding transcription factor binding / DNA-binding transcription factor activity, RNA polymerase II-specific / calmodulin binding / nuclear speck / RNA polymerase II cis-regulatory region sequence-specific DNA binding / DNA repair / positive regulation of gene expression / chromatin / nucleolus / positive regulation of DNA-templated transcription / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / DNA binding / nucleoplasm / nucleus / metal ion binding / cytoplasm
Similarity search - Function
Permuted single zf-CXXC unit / Demeter, RRM-fold domain / DNA glycosylase, plant / RRM in Demeter / Permuted single zf-CXXC unit / Transcription factor SRY / : / Endonuclease III-like, iron-sulphur cluster loop motif / FES / Helix-hairpin-helix, base-excision DNA repair, C-terminal ...Permuted single zf-CXXC unit / Demeter, RRM-fold domain / DNA glycosylase, plant / RRM in Demeter / Permuted single zf-CXXC unit / Transcription factor SRY / : / Endonuclease III-like, iron-sulphur cluster loop motif / FES / Helix-hairpin-helix, base-excision DNA repair, C-terminal / HhH-GPD domain / endonuclease III / DNA glycosylase / HMG (high mobility group) box / HMG boxes A and B DNA-binding domains profile. / high mobility group / High mobility group box domain / High mobility group box domain superfamily
Similarity search - Domain/homology
Sex-determining region Y protein / DNA glycosylase/AP lyase ROS1
Similarity search - Component
Biological speciesHomo sapiens (human) / Arabidopsis thaliana (thale cress)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsDu X / Du J
Funding support1 items
OrganizationGrant numberCountry
Other government
CitationJournal: Nat Plants / Year: 2023
Title: Molecular basis of the plant ROS1-mediated active DNA demethylation.
Authors: Xuan Du / Zhenlin Yang / Guohui Xie / Changshi Wang / Laixing Zhang / Kaige Yan / Maojun Yang / Sisi Li / Jian-Kang Zhu / Jiamu Du /
Abstract: Active DNA demethylation plays a crucial role in eukaryotic gene imprinting and antagonizing DNA methylation. The plant-specific REPRESSOR OF SILENCING 1/DEMETER (ROS1/DME) family of enzymes directly ...Active DNA demethylation plays a crucial role in eukaryotic gene imprinting and antagonizing DNA methylation. The plant-specific REPRESSOR OF SILENCING 1/DEMETER (ROS1/DME) family of enzymes directly excise 5-methyl-cytosine (5mC), representing an efficient DNA demethylation pathway distinct from that of animals. Here, we report the cryo-electron microscopy structures of an Arabidopsis ROS1 catalytic fragment in complex with substrate DNA, mismatch DNA and reaction intermediate, respectively. The substrate 5mC is flipped-out from the DNA duplex and subsequently recognized by the ROS1 base-binding pocket through hydrophobic and hydrogen-bonding interactions towards the 5-methyl group and Watson-Crick edge respectively, while the different protonation states of the bases determine the substrate preference for 5mC over T:G mismatch. Together with the structure of the reaction intermediate complex, our structural and biochemical studies revealed the molecular basis for substrate specificity, as well as the reaction mechanism underlying 5mC demethylation by the ROS1/DME family of plant-specific DNA demethylases.
History
DepositionJul 14, 2022-
Header (metadata) releaseNov 30, 2022-
Map releaseNov 30, 2022-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33836.png

Downloads & links

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Map

FileDownload / File: emd_33836.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.67 Å/pix.
x 400 pix.
= 268. Å		0.67 Å/pix.
x 400 pix.
= 268. Å		0.67 Å/pix.
x 400 pix.
= 268. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.67 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00675
Minimum - Maximum-0.02384263 - 0.062653325
Average (Standard dev.)0.000027355658 (±0.00063778035)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 268.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_33836_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_33836_half_map_2.map
Projections & Slices
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Sample components

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Entire : covalent-linked reaction intermediate of Arabidopsis ROS1 with dsDNA

EntireName: covalent-linked reaction intermediate of Arabidopsis ROS1 with dsDNA
Components
  • Complex: covalent-linked reaction intermediate of Arabidopsis ROS1 with dsDNA
    • Complex: ROS1
      • Protein or peptide: Sex-determining region Y protein,DNA glycosylase/AP lyase ROS1
    • Complex: dsDNA
      • DNA: DNA (40-MER)
      • DNA: DNA (40-MER)
  • Ligand: IRON/SULFUR CLUSTER

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Supramolecule #1: covalent-linked reaction intermediate of Arabidopsis ROS1 with dsDNA

SupramoleculeName: covalent-linked reaction intermediate of Arabidopsis ROS1 with dsDNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: ROS1

SupramoleculeName: ROS1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: dsDNA

SupramoleculeName: dsDNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Arabidopsis thaliana (thale cress)

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Macromolecule #1: Sex-determining region Y protein,DNA glycosylase/AP lyase ROS1

MacromoleculeName: Sex-determining region Y protein,DNA glycosylase/AP lyase ROS1
type: protein_or_peptide / ID: 1
Details: Fusion protein of residues 56 to 130 of database UNP Q05066, Linker, residues 511 to 1393 of database UNP Q9SJQ6, with deletion of residues 665-835 and 1074-1109.
Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-(apurinic or apyrimidinic site) lyase
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 86.261844 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: SVQDRVKRPM NAFIVWSRDQ RRKMALENPR MRNSEISKQL GYQWKMLTEA EKWPFFQEAQ KLQAMHREKY PNYKYRKGGS SAGAIVPVT PVKKPRPRPK VDLDDETDRV WKLLLENINS EGVDGSDEQK AKWWEEERNV FRGRADSFIA RMHLVQGDRR F TPWKGSVV ...String:
SVQDRVKRPM NAFIVWSRDQ RRKMALENPR MRNSEISKQL GYQWKMLTEA EKWPFFQEAQ KLQAMHREKY PNYKYRKGGS SAGAIVPVT PVKKPRPRPK VDLDDETDRV WKLLLENINS EGVDGSDEQK AKWWEEERNV FRGRADSFIA RMHLVQGDRR F TPWKGSVV DSVVGVFLTQ NVSDHLSSSA FMSLASQFPV PFVPSSNFDA GTSSMPSIQI TYLDSEETMS SPPDHNHSCQ KP TLKEKGK KVLKEEKKAF DWDCLRREAQ ARAGIREKTR STMDTVDWKA IRAADVKEVA ETIKSRGMNH KLAERIQGFL DRL VNDHGS IDLEWLRDVP PDKAKEYLLS FNGLGLKSVE CVRLLTLHHL AFPVDTNVGR IAVRLGWVPL QPLPESLQLH LLEM YPMLE SIQKYLWPRL CKLDQKTLYE LHYQMITFGK VFCTKSKPNC NACPMKGECR HFASAFASAR LALPSTEKTC CEPII EEPA SPEPETAEVS IADIEEAFFE DPEEIPTIRL NMDAFTSNLK KIMEHNKELQ DGNMSSALVA LTAETASLPM PKLKNI SQL RTEHRVYELP DEHPLLAQLE KREPDDPCSY LLAIWTPGET ADSIQPSVST CIFQANGMLC DEETCFSCNS IKETRSQ IV RGTILIPCRT AMRGSFPLNG TYFQVNEVFA DHASSLNPIN VPRELIWELP RRTVYFGTSV PTIFKGLSTE KIQACFWK G YVCVRGFDRK TRGPKPLIAR LHFPASKLKG QQANLA

UniProtKB: Sex-determining region Y protein, DNA glycosylase/AP lyase ROS1, DNA glycosylase/AP lyase ROS1, DNA glycosylase/AP lyase ROS1

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Macromolecule #2: DNA (40-MER)

MacromoleculeName: DNA (40-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 12.345953 KDa
SequenceString:
(DT)(DA)(DG)(DC)(DT)(DG)(DA)(DG)(DT)(DG) (DA)(DT)(DC)(DC)(DG)(DG)(DA)(DG)(DT)(DC) (DT)(DG)(DA)(DA)(DA)(DC)(DA)(DA)(DT) (DG)(DC)(DA)(DT)(DC)(DA)(DC)(DA)(DT)(DG) (DC)

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Macromolecule #3: DNA (40-MER)

MacromoleculeName: DNA (40-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 12.171816 KDa
SequenceString:
(DG)(DC)(DA)(DT)(DG)(DT)(DG)(DA)(DT)(DG) (DC)(DA)(DT)(DT)(DG)(DT)(DT)(DT)(DC)(DA) (DG)(DA)(DC)(DT)(DC)(PED)(DG)(DG)(DA) (DT)(DC)(DA)(DC)(DT)(DC)(DA)(DG)(DC)(DT) (DA)

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Macromolecule #4: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 4 / Number of copies: 1 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER

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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

Concentration0.7 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 %

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 1.3467 sec. / Average electron dose: 1.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: OTHER
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
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: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 84039
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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