+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-24635 | |||||||||
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Title | Arabidopsis RNA-dependent RNA polymerase 2 | |||||||||
Map data | EM map for RNA-dependent RNA polymerase 2 | |||||||||
Sample |
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Keywords | siRNA / epigenetics / plants / RNA polymerase / gene silencing / TRANSCRIPTION | |||||||||
Function / homology | Function and homology information siRNA-mediated long-distance post-transcriptional gene silencing / siRNA transcription / DNA/RNA hybrid binding / siRNA processing / defense response to fungus / single-stranded RNA binding / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / nucleolus / nucleoplasm ...siRNA-mediated long-distance post-transcriptional gene silencing / siRNA transcription / DNA/RNA hybrid binding / siRNA processing / defense response to fungus / single-stranded RNA binding / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / nucleolus / nucleoplasm / nucleus / metal ion binding Similarity search - Function | |||||||||
Biological species | Arabidopsis thaliana (thale cress) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.57 Å | |||||||||
Authors | Fukudome A / Pikaard CS | |||||||||
Funding support | United States, 2 items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2021 Title: Structure and RNA template requirements of RNA-DEPENDENT RNA POLYMERASE 2. Authors: Akihito Fukudome / Jasleen Singh / Vibhor Mishra / Eswar Reddem / Francisco Martinez-Marquez / Sabine Wenzel / Rui Yan / Momoko Shiozaki / Zhiheng Yu / Joseph Che-Yen Wang / Yuichiro Takagi / Craig S Pikaard / Abstract: RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In , RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA ...RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In , RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest, induced by the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The double-stranded RNAs are then released from the Pol IV-RDR2 complex and diced into short-interfering RNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 Å, determined by cryoelectron microscopy. The N-terminal region contains an RNA-recognition motif adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1 to 2 nt internal to the 3' ends of its templates and can transcribe the RNA of an RNA/DNA hybrid, provided that 9 or more nucleotides are unpaired at the RNA's 3' end. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3' end occurs as the DNA template and nontemplate strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3' end as the DNA strands reanneal, allowing RDR2 to engage the RNA and synthesize the complementary strand. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_24635.map.gz | 437.3 MB | EMDB map data format | |
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Header (meta data) | emd-24635-v30.xml emd-24635.xml | 18.9 KB 18.9 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_24635_fsc.xml | 17.8 KB | Display | FSC data file |
Images | emd_24635.png | 113.7 KB | ||
Filedesc metadata | emd-24635.cif.gz | 6.3 KB | ||
Others | emd_24635_half_map_1.map.gz emd_24635_half_map_2.map.gz | 474.7 MB 474.7 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-24635 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-24635 | HTTPS FTP |
-Validation report
Summary document | emd_24635_validation.pdf.gz | 871.5 KB | Display | EMDB validaton report |
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Full document | emd_24635_full_validation.pdf.gz | 871 KB | Display | |
Data in XML | emd_24635_validation.xml.gz | 26.9 KB | Display | |
Data in CIF | emd_24635_validation.cif.gz | 35 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-24635 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-24635 | HTTPS FTP |
-Related structure data
Related structure data | 7rqsMC 7rozC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_24635.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | EM map for RNA-dependent RNA polymerase 2 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.422 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Half map: Half map1
File | emd_24635_half_map_1.map | ||||||||||||
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Annotation | Half map1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map2
File | emd_24635_half_map_2.map | ||||||||||||
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Annotation | Half map2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : RNA-dependent RNA polymerase 2
Entire | Name: RNA-dependent RNA polymerase 2 |
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Components |
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-Supramolecule #1: RNA-dependent RNA polymerase 2
Supramolecule | Name: RNA-dependent RNA polymerase 2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 |
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Source (natural) | Organism: Arabidopsis thaliana (thale cress) |
Molecular weight | Theoretical: 129 KDa |
-Macromolecule #1: RNA-dependent RNA polymerase 2
Macromolecule | Name: RNA-dependent RNA polymerase 2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed RNA polymerase |
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Source (natural) | Organism: Arabidopsis thaliana (thale cress) |
Molecular weight | Theoretical: 133.619828 KDa |
Recombinant expression | Organism: Insect cell expression vector pTIE1 (others) |
Sequence | String: MVSETTTNRS TVKISNVPQT IVADELLRFL ELHLGEDTVF ALEIPTTRDN WKPRDFARVQ FTTLEVKSRA QLLSSQSKLL FKTHNLRLS EAYDDIIPRP VDPRKRLDDI VLTVGFPESD EKRFCALEKW DGVRCWILTE KRRVEFWVWE SGDCYKIEVR F EDIIETLS ...String: MVSETTTNRS TVKISNVPQT IVADELLRFL ELHLGEDTVF ALEIPTTRDN WKPRDFARVQ FTTLEVKSRA QLLSSQSKLL FKTHNLRLS EAYDDIIPRP VDPRKRLDDI VLTVGFPESD EKRFCALEKW DGVRCWILTE KRRVEFWVWE SGDCYKIEVR F EDIIETLS CCVNGDASEI DAFLLKLKYG PKVFKRVTVH IATKFKSDRY RFCKEDFDFM WIRTTDFSGS KSIGTSTCFC LE VHNGSTM LDIFSGLPYY REDTLSLTYV DGKTFASAAQ IVPLLNAAIL GLEFPYEILF QLNALVHAQK ISLFAASDME LIK ILRGMS LETALVILKK LHQQSSICYD PVFFVKTQMQ SVVKKMKHSP ASAYKRLTEQ NIMSCQRAYV TPSKIYLLGP ELET ANYVV KNFAEHVSDF MRVTFVEEDW SKLPANALSV NSKEGYFVKP SRTNIYNRVL SILGEGITVG PKRFEFLAFS ASQLR GNSV WMFASNEKVK AEDIREWMGC FRKIRSISKC AARMGQLFSA SRQTLIVRAQ DVEQIPDIEV TTDGADYCFS DGIGKI SLA FAKQVAQKCG LSHVPSAFQI RYGGYKGVIA VDRSSFRKLS LRDSMLKFDS NNRMLNVTRW TESMPCFLNR EIICLLS TL GIEDAMFEAM QAVHLSMLGN MLEDRDAALN VLQKLSGENS KNLLVKMLLQ GYAPSSEPYL SMMLRVHHES QLSELKSR C RILVPKGRIL IGCMDEMGIL EYGQVYVRVT LTKAELKSRD QSYFRKIDEE TSVVIGKVVV TKNPCLHPGD IRVLDAIYE VHFEEKGYLD CIIFPQKGER PHPNECSGGD LDGDQFFVSW DEKIIPSEMD PPMDYAGSRP RLMDHDVTLE EIHKFFVDYM ISDTLGVIS TAHLVHADRD PEKARSQKCL ELANLHSRAV DFAKTGAPAE MPYALKPREF PDFLERFEKP TYISESVFGK L YRAVKSSL AQRKPEAESE DTVAYDVTLE EAGFESFIET AKAHRDMYGE KLTSLMIYYG AANEEEILTG ILKTKEMYLA RD NRRYGDM KDRITLSVKD LHKEAMGWFE KSCEDEQQKK KLASAWYYVT YNPNHRDEKL TFLSFPWIVG DVLLDIKAEN AQR QSVEEK TSGLVSIKLE NLYFQGSAWS HPQFEKGGGS GGGSGGSAWS HPQFEK UniProtKB: RNA-dependent RNA polymerase 2 |
-Macromolecule #2: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-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: 7.6 |
Grid | Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: GOLD / Support film - topology: HOLEY |
Vitrification | Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV |
Details | The sample was mono disperse. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | #0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / #0 - Number grids imaged: 1 / #0 - Number real images: 6417 / #0 - Average exposure time: 4.0 sec. / #0 - Average electron dose: 60.0 e/Å2 / #0 - Details: Non-tilted dataset / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / #1 - Number grids imaged: 1 / #1 - Number real images: 4545 / #1 - Average exposure time: 4.0 sec. / #1 - Average electron dose: 60.0 e/Å2 / #1 - Details: 30 degree titled dataset |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 59242 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -2.0 µm / Nominal defocus min: -0.8 µm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: AB INITIO MODEL / Overall B value: 173.2 |
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Output model | PDB-7rqs: |