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- PDB-7yhq: CryoEM structure of Arabidopsis ROS1 in complex with a covalent-l... -

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Basic information

Entry
Database: PDB / ID: 7yhq
TitleCryoEM structure of Arabidopsis ROS1 in complex with a covalent-linked reaction intermediate at 3.9 Angstroms resolution
Components
  • (DNA (40-MER)) x 2
  • Sex-determining region Y protein,DNA glycosylase/AP lyase ROS1
KeywordsDNA BINDING PROTEIN/DNA / ROS1 / DNA glycosylase / DNA demethylation / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
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-(apurinic or apyrimidinic site) endonuclease activity ...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-(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 / neuron differentiation / brain development / 4 iron, 4 sulfur cluster binding / DNA-binding transcription activator activity, RNA polymerase II-specific / DNA-binding transcription factor binding / 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 / 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 / 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
IRON/SULFUR CLUSTER / DNA / DNA (> 10) / Sex-determining region Y protein / DNA glycosylase/AP lyase ROS1
Similarity search - Component
Biological speciesHomo sapiens (human)
Arabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsDu, X. / Du, J.
Funding support1items
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, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 30, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 8, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Sex-determining region Y protein,DNA glycosylase/AP lyase ROS1
B: DNA (40-MER)
C: DNA (40-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)111,1314
Polymers110,7803
Non-polymers3521
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Sex-determining region Y protein,DNA glycosylase/AP lyase ROS1 / Testis-determining factor / DEMETER-like protein 1 / Protein REPRESSOR OF SILENCING 1 / Protein ROS1


Mass: 86261.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
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.
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Arabidopsis thaliana (thale cress)
Gene: SRY, TDF, ROS1, DML1, At2g36490, F1O11.12
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q05066, UniProt: Q9SJQ6, DNA-(apurinic or apyrimidinic site) lyase
#2: DNA chain DNA (40-MER)


Mass: 12345.953 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Production host: Escherichia coli (E. coli)
#3: DNA chain DNA (40-MER)


Mass: 12171.816 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Production host: Escherichia coli (E. coli)
#4: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1covalent-linked reaction intermediate of Arabidopsis ROS1 with dsDNACOMPLEX#1-#30RECOMBINANT
2ROS1COMPLEX#11RECOMBINANT
3dsDNACOMPLEX#2-#31RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
22Arabidopsis thaliana (thale cress)3702
33Arabidopsis thaliana (thale cress)3702
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
23Escherichia coli (E. coli)562
Buffer solutionpH: 8
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: OTHER / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingAverage exposure time: 1.3467 sec. / Electron dose: 1.2 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 84039 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0064254
ELECTRON MICROSCOPYf_angle_d0.9425944
ELECTRON MICROSCOPYf_dihedral_angle_d23.6681624
ELECTRON MICROSCOPYf_chiral_restr0.051655
ELECTRON MICROSCOPYf_plane_restr0.007604

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