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Yorodumi- EMDB-28113: Cryo-EM structure of consensus elemental paused elongation comple... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-28113 | |||||||||
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Title | Cryo-EM structure of consensus elemental paused elongation complex with an unfolded TL | |||||||||
Map data | a postprocessed map | |||||||||
Sample |
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Keywords | RNA polymerase / cryo-EM structure / elemental pause / Escherichia coli / transcription / TRANSFERASE-DNA-RNA complex / transcriptional regulation / transcriptional pausing | |||||||||
Function / homology | Function and homology information DNA-directed RNA polymerase complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm Similarity search - Function | |||||||||
Biological species | Escherichia coli (E. coli) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.8 Å | |||||||||
Authors | Kang JY / Chen J / Llewellyn E / Landick R / Darst SA | |||||||||
Funding support | United States, 2 items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2023 Title: An ensemble of interconverting conformations of the elemental paused transcription complex creates regulatory options. Authors: Jin Young Kang / Tatiana V Mishanina / Yu Bao / James Chen / Eliza Llewellyn / James Liu / Seth A Darst / Robert Landick / Abstract: Transcriptional pausing underpins the regulation of cellular RNA synthesis, but its mechanism remains incompletely understood. Sequence-specific interactions of DNA and RNA with the dynamic, ...Transcriptional pausing underpins the regulation of cellular RNA synthesis, but its mechanism remains incompletely understood. Sequence-specific interactions of DNA and RNA with the dynamic, multidomain RNA polymerase (RNAP) trigger reversible conformational changes at pause sites that temporarily interrupt the nucleotide addition cycle. These interactions initially rearrange the elongation complex (EC) into an elemental paused EC (ePEC). ePECs can form longer-lived PECs by further rearrangements or interactions of diffusible regulators. For both bacterial and mammalian RNAPs, a half-translocated state in which the next DNA template base fails to load into the active site appears central to the ePEC. Some RNAPs also swivel interconnected modules that may stabilize the ePEC. However, it is unclear whether swiveling and half-translocation are requisite features of a single ePEC state or if multiple ePEC states exist. Here, we use cryo-electron microscopy (cryo-EM) analysis of ePECs with different RNA-DNA sequences combined with biochemical probes of ePEC structure to define an interconverting ensemble of ePEC states. ePECs occupy either pre- or half-translocated states but do not always swivel, indicating that difficulty in forming the posttranslocated state at certain RNA-DNA sequences may be the essence of the ePEC. The existence of multiple ePEC conformations has broad implications for transcriptional regulation. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_28113.map.gz | 60 MB | EMDB map data format | |
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Header (meta data) | emd-28113-v30.xml emd-28113.xml | 32.8 KB 32.8 KB | Display Display | EMDB header |
Images | emd_28113.png | 151.9 KB | ||
Filedesc metadata | emd-28113.cif.gz | 9.4 KB | ||
Others | emd_28113_additional_1.map.gz emd_28113_half_map_1.map.gz emd_28113_half_map_2.map.gz | 51.4 MB 49.7 MB 49.7 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-28113 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-28113 | HTTPS FTP |
-Validation report
Summary document | emd_28113_validation.pdf.gz | 990.8 KB | Display | EMDB validaton report |
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Full document | emd_28113_full_validation.pdf.gz | 990.4 KB | Display | |
Data in XML | emd_28113_validation.xml.gz | 12 KB | Display | |
Data in CIF | emd_28113_validation.cif.gz | 13.7 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-28113 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-28113 | HTTPS FTP |
-Related structure data
Related structure data | 8egbMC 8eg7C 8eg8C 8eh8C 8eh9C 8ehaC 8ehfC 8ehiC C: citing same article (ref.) M: atomic model generated by this map |
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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_28113.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | a postprocessed map | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.3 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: a local-filtered map
File | emd_28113_additional_1.map | ||||||||||||
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Annotation | a local-filtered map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: a second half map
File | emd_28113_half_map_1.map | ||||||||||||
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Annotation | a second half map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: a first half mpa
File | emd_28113_half_map_2.map | ||||||||||||
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Annotation | a first half mpa | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
+Entire : consensus elemental paused elongation complex
+Supramolecule #1: consensus elemental paused elongation complex
+Macromolecule #1: non-template DNA
+Macromolecule #2: template DNA
+Macromolecule #3: RNA
+Macromolecule #4: DNA-directed RNA polymerase subunit alpha
+Macromolecule #5: DNA-directed RNA polymerase subunit beta
+Macromolecule #6: DNA-directed RNA polymerase subunit beta'
+Macromolecule #7: DNA-directed RNA polymerase subunit omega
+Macromolecule #8: CHAPSO
+Macromolecule #9: MAGNESIUM ION
+Macromolecule #10: ZINC ION
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 8 mg/mL | |||||||||||||||
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Buffer | pH: 8 Component:
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Grid | Model: C-flat-1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 22 K / Instrument: FEI VITROBOT MARK IV / Details: blot force 0, blot time 3 s. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 2525 / Average exposure time: 10.0 sec. / Average electron dose: 47.34 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model |
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Refinement | Space: REAL / Protocol: RIGID BODY FIT | ||||||||||||
Output model | PDB-8egb: |