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Yorodumi- EMDB-33835: CryoEM structure of Arabidopsis ROS1 in complex with 5mC-dsDNA at... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-33835 | |||||||||
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Title | CryoEM structure of Arabidopsis ROS1 in complex with 5mC-dsDNA at 3.1 Angstroms resolution | |||||||||
Map data | ||||||||||
Sample |
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Keywords | 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 / defense response to fungus / DNA-(apurinic or apyrimidinic site) endonuclease activity / class I 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 / 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 / brain development / base-excision repair / neuron differentiation / 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 | |||||||||
Biological species | Homo sapiens (human) / Arabidopsis thaliana (thale cress) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.1 Å | |||||||||
Authors | Du X / Du J | |||||||||
Funding support | 1 items
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_33835.map.gz | 15.2 MB | EMDB map data format | |
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Header (meta data) | emd-33835-v30.xml emd-33835.xml | 18 KB 18 KB | Display Display | EMDB header |
Images | emd_33835.png | 66.9 KB | ||
Filedesc metadata | emd-33835.cif.gz | 6.3 KB | ||
Others | emd_33835_half_map_1.map.gz emd_33835_half_map_2.map.gz | 195 MB 195 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-33835 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-33835 | HTTPS FTP |
-Validation report
Summary document | emd_33835_validation.pdf.gz | 769 KB | Display | EMDB validaton report |
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Full document | emd_33835_full_validation.pdf.gz | 768.6 KB | Display | |
Data in XML | emd_33835_validation.xml.gz | 15.8 KB | Display | |
Data in CIF | emd_33835_validation.cif.gz | 18.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-33835 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-33835 | HTTPS FTP |
-Related structure data
Related structure data | 7yhpMC 7yhoC 7yhqC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_33835.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.668 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #2
File | emd_33835_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_33835_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : ROS1-5mC dsDNA complex
Entire | Name: ROS1-5mC dsDNA complex |
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Components |
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-Supramolecule #1: ROS1-5mC dsDNA complex
Supramolecule | Name: ROS1-5mC dsDNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 |
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-Supramolecule #2: ROS1
Supramolecule | Name: ROS1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: Homo sapiens (human) |
-Supramolecule #3: 5mC dsDNA
Supramolecule | Name: 5mC dsDNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3 |
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Source (natural) | Organism: Arabidopsis thaliana (thale cress) |
-Macromolecule #1: Sex-determining region Y protein,REPRESSOR OF SILENCING 1,DNA gly...
Macromolecule | Name: Sex-determining region Y protein,REPRESSOR OF SILENCING 1,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 |
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Source (natural) | Organism: Arabidopsis thaliana (thale cress) |
Molecular weight | Theoretical: 86.260859 KDa |
Recombinant expression | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Sequence | String: 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 UniProtKB: Sex-determining region Y protein, DNA glycosylase/AP lyase ROS1, DNA glycosylase/AP lyase ROS1, DNA glycosylase/AP lyase ROS1 |
-Macromolecule #2: DNA (40-MER)
Macromolecule | Name: DNA (40-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: Arabidopsis thaliana (thale cress) |
Molecular weight | Theoretical: 12.336939 KDa |
Sequence | String: (DT)(DA)(DG)(DC)(DT)(DG)(DT)(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) |
-Macromolecule #3: DNA (40-MER)
Macromolecule | Name: DNA (40-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: Arabidopsis thaliana (thale cress) |
Molecular weight | Theoretical: 12.292913 KDa |
Sequence | String: (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)(5CM)(DG)(DG)(DA) (DT)(DC)(DA)(DC)(DT)(DC)(DA)(DG)(DC)(DT) (DA) |
-Macromolecule #4: IRON/SULFUR CLUSTER
Macromolecule | Name: IRON/SULFUR CLUSTER / type: ligand / ID: 4 / Number of copies: 1 / Formula: SF4 |
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Molecular weight | Theoretical: 351.64 Da |
Chemical component information | ChemComp-FS1: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.7 mg/mL |
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Buffer | pH: 8 |
Grid | Model: Quantifoil R0.6/1 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average exposure time: 1.3467 sec. / Average electron dose: 1.2 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: OTHER |
Electron optics | Illumination 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 |
-Image processing
Startup model | Type of model: NONE |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 116523 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |