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- EMDB-33832: CryoEM structure of Arabidopsis ROS1 in complex with TG mismatch ... -

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Basic information

Entry
Database: EMDB / ID: EMD-33832
TitleCryoEM structure of Arabidopsis ROS1 in complex with TG mismatch dsDNA at 3.3 Angstroms resolution
Map data
Sample
  • Complex: ROS1-TG mismatch dsDNA complex
    • Complex: ROS1
      • Protein or peptide: Sex-determining region Y protein,DNA glycosylase/AP lyase ROS1
    • Complex: TG mismatch dsDNA
      • DNA: DNA (40-MER)
      • DNA: DNA (40-MER)
  • Ligand: IRON/SULFUR CLUSTERIron–sulfur cluster
Function / homology
Function and homology information


DNA demethylase activity / positive regulation of male gonad development / Transcriptional regulation of testis differentiation / sex differentiation / DNA N-glycosylase activity / male sex determination / : / : / anatomical structure morphogenesis / defense response to fungus ...DNA demethylase activity / positive regulation of male gonad development / Transcriptional regulation of testis differentiation / sex differentiation / DNA N-glycosylase activity / male sex determination / : / : / anatomical structure morphogenesis / defense response to fungus / class I DNA-(apurinic or apyrimidinic site) endonuclease activity / DNA-(apurinic or apyrimidinic site) endonuclease activity / DNA-(apurinic or apyrimidinic site) lyase / Deactivation of the beta-catenin transactivating complex / base-excision repair / 4 iron, 4 sulfur cluster binding / DNA-binding transcription factor binding / cell differentiation / calmodulin binding / DNA-binding transcription factor activity, RNA polymerase II-specific / nuclear speck / RNA polymerase II cis-regulatory region sequence-specific DNA binding / DNA repair / chromatin / positive regulation of gene expression / 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 / metal ion binding / nucleus / 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 / HhH-GPD domain ...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: 3.3 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-
UpdateMar 8, 2023-
Current statusMar 8, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33832.png

Downloads & links

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Map

FileDownload / File: emd_33832.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.67 Å
Density
Contour LevelBy AUTHOR: 0.00654
Minimum - Maximum-0.032055702 - 0.054920956
Average (Standard dev.)3.1883672e-05 (±0.0006665996)
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_33832_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_33832_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 : ROS1-TG mismatch dsDNA complex

EntireName: ROS1-TG mismatch dsDNA complex
Components
  • Complex: ROS1-TG mismatch dsDNA complex
    • Complex: ROS1
      • Protein or peptide: Sex-determining region Y protein,DNA glycosylase/AP lyase ROS1
    • Complex: TG mismatch dsDNA
      • DNA: DNA (40-MER)
      • DNA: DNA (40-MER)
  • Ligand: IRON/SULFUR CLUSTERIron–sulfur cluster

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Supramolecule #1: ROS1-TG mismatch dsDNA complex

SupramoleculeName: ROS1-TG mismatch dsDNA complex / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: ROS1

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

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Supramolecule #3: TG mismatch dsDNA

SupramoleculeName: TG mismatch dsDNA / type: complex / ID: 3 / Chimera: Yes / 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: 86.260859 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 AFPVNTNVGR 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

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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: 12.39499 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)(DG)(DA) (DG)

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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: 12.244859 KDa
SequenceString:
(DC)(DT)(DC)(DT)(DG)(DT)(DG)(DA)(DT)(DG) (DC)(DA)(DT)(DT)(DG)(DT)(DT)(DT)(DC)(DA) (DG)(DA)(DC)(DT)(DC)(DT)(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 / 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
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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 1.3467 sec. / Average electron dose: 1.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 111356

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