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Yorodumi- EMDB-1803: Conformational flexibility of RNA polymerase III during transcrip... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-1803 | |||||||||
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Title | Conformational flexibility of RNA polymerase III during transcriptional elongation | |||||||||
Map data | RNA Polymerase III elongation complex | |||||||||
Sample |
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Keywords | Transcription / RNA polymerase III / elongation complex / transcription initiation / transcription elongation / transcription termination | |||||||||
Biological species | synthetic construct (others) / Saccharomyces cerevisiae (brewer's yeast) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 16.5 Å | |||||||||
Authors | Fernandez-Tornero C / Bottcher B / Rashid UJ / Steuerwald U / Florchinger B / Devos DP / Lindner D / Muller CW | |||||||||
Citation | Journal: EMBO J / Year: 2010 Title: Conformational flexibility of RNA polymerase III during transcriptional elongation. Authors: Carlos Fernández-Tornero / Bettina Böttcher / Umar Jan Rashid / Ulrich Steuerwald / Beate Flörchinger / Damien P Devos / Doris Lindner / Christoph W Müller / Abstract: RNA polymerase (Pol) III is responsible for the transcription of genes encoding small RNAs, including tRNA, 5S rRNA and U6 RNA. Here, we report the electron cryomicroscopy structures of yeast Pol III ...RNA polymerase (Pol) III is responsible for the transcription of genes encoding small RNAs, including tRNA, 5S rRNA and U6 RNA. Here, we report the electron cryomicroscopy structures of yeast Pol III at 9.9 Å resolution and its elongation complex at 16.5 Å resolution. Particle sub-classification reveals prominent EM densities for the two Pol III-specific subcomplexes, C31/C82/C34 and C37/C53, that can be interpreted using homology models. While the winged-helix-containing C31/C82/C34 subcomplex initiates transcription from one side of the DNA-binding cleft, the C37/C53 subcomplex accesses the transcription bubble from the opposite side of this cleft. The transcribing Pol III enzyme structure not only shows the complete incoming DNA duplex, but also reveals the exit path of newly synthesized RNA. During transcriptional elongation, the Pol III-specific subcomplexes tightly enclose the incoming DNA duplex, which likely increases processivity and provides structural insights into the conformational switch between Pol III-mediated initiation and elongation. | |||||||||
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_1803.map.gz | 2.5 MB | EMDB map data format | |
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Header (meta data) | emd-1803-v30.xml emd-1803.xml | 10.6 KB 10.6 KB | Display Display | EMDB header |
Images | 1803.png | 117.7 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-1803 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-1803 | HTTPS FTP |
-Validation report
Summary document | emd_1803_validation.pdf.gz | 203.9 KB | Display | EMDB validaton report |
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Full document | emd_1803_full_validation.pdf.gz | 203 KB | Display | |
Data in XML | emd_1803_validation.xml.gz | 5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1803 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1803 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_1803.map.gz / Format: CCP4 / Size: 2.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | RNA Polymerase III elongation complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.8 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : RNA Polymerase III elongation complex
Entire | Name: RNA Polymerase III elongation complex |
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Components |
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-Supramolecule #1000: RNA Polymerase III elongation complex
Supramolecule | Name: RNA Polymerase III elongation complex / type: sample / ID: 1000 / Details: Sample was monodisperse / Oligomeric state: Transcription bubble, RNA Pol III complex / Number unique components: 2 |
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Molecular weight | Experimental: 700 KDa / Theoretical: 700 KDa / Method: Native mass spectrometry |
-Macromolecule #1: Transcription bubble
Macromolecule | Name: Transcription bubble / type: other / ID: 1 Details: An artificial transcription bubble was prepared as described (Kettenberger et al., 2004) and was added in five molar excess to purified Pol III. Unbound transcription bubble was subsequently ...Details: An artificial transcription bubble was prepared as described (Kettenberger et al., 2004) and was added in five molar excess to purified Pol III. Unbound transcription bubble was subsequently removed using size exclusion chromatography Classification: DNA/RNA / Structure: OTHER / Synthetic?: Yes |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 30 KDa |
-Macromolecule #2: RNA Polymerase III
Macromolecule | Name: RNA Polymerase III / type: protein_or_peptide / ID: 2 / Name.synonym: Pol III / Oligomeric state: One complex of 17 different polypeptides / Recombinant expression: No |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: Nuclear |
Molecular weight | Experimental: 700 KDa / Theoretical: 700 KDa |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.5 mg/mL |
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Buffer | pH: 7.5 Details: 10 mM Tris pH 7.5, 100 mM ammonium sulphate, 10 mM DTT. |
Grid | Details: Lacey carbon film |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: HOMEMADE PLUNGER Details: Vitrification instrument: Controlled environment, self-built Method: Blot for 15 s in controlled environment chamber before plunging |
-Electron microscopy
Microscope | FEI/PHILIPS CM200FEG |
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Temperature | Average: 95 K |
Alignment procedure | Legacy - Astigmatism: Astigmatism was corrected at 200,000 times magnification |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm / Number real images: 39 / Bits/pixel: 8 |
Tilt angle min | 0 |
Tilt angle max | 0 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN |
-Image processing
Details | ca. 23892 particles were sorted into four groups according to the compositional differences. The reconstruction shows the most complete species and represents ca. 49% of the particle population. |
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CTF correction | Details: Each particle, phase flipped |
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER Details: Maps were calculated from defocus groups,and added. Number images used: 11700 |