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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-8586 | |||||||||
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Title | CryoEM structure of E.coli RNA polymerase elongation complex | |||||||||
![]() | CryoEM structure of crosslinked E.coli RNA polymerase elongation complex | |||||||||
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Function / homology | ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() | |||||||||
Method | ![]() ![]() | |||||||||
![]() | Kang JY / Darst SA | |||||||||
![]() | ![]() Title: Structural basis of transcription arrest by coliphage HK022 Nun in an RNA polymerase elongation complex. Authors: Jin Young Kang / Paul Dominic B Olinares / James Chen / Elizabeth A Campbell / Arkady Mustaev / Brian T Chait / Max E Gottesman / Seth A Darst / ![]() Abstract: Coliphage HK022 Nun blocks superinfection by coliphage λ by stalling RNA polymerase (RNAP) translocation specifically on λ DNA. To provide a structural framework to understand how Nun blocks RNAP ...Coliphage HK022 Nun blocks superinfection by coliphage λ by stalling RNA polymerase (RNAP) translocation specifically on λ DNA. To provide a structural framework to understand how Nun blocks RNAP translocation, we determined structures of RNAP ternary elongation complexes (TECs) with and without Nun by single-particle cryo-electron microscopy. Nun fits tightly into the TEC by taking advantage of gaps between the RNAP and the nucleic acids. The C-terminal segment of Nun interacts with the RNAP β and β' subunits inside the RNAP active site cleft as well as with nearly every element of the nucleic acid scaffold, essentially crosslinking the RNAP and the nucleic acids to prevent translocation, a mechanism supported by the effects of Nun amino acid substitutions. The nature of Nun interactions inside the RNAP active site cleft suggests that RNAP clamp opening is required for Nun to establish its interactions, explaining why Nun acts on paused TECs. | |||||||||
History |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 59.9 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 23.7 KB 23.7 KB | Display Display | ![]() |
Images | ![]() | 66.2 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6alhMC ![]() 8584C ![]() 8585C ![]() 6alfC ![]() 6algC ![]() 5upc C: citing same article ( M: atomic model generated by this map |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | CryoEM structure of crosslinked E.coli RNA polymerase elongation complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.3 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
+Entire : E.coli RNA polymerase ternary elongation complex
+Supramolecule #1: E.coli RNA polymerase ternary elongation complex
+Macromolecule #1: DNA (29-MER)
+Macromolecule #2: DNA (29-MER)
+Macromolecule #3: RNA (5'-R(*GP*CP*A*UP*UP*CP*AP*AP*AP*GP*CP*GP*GP*AP*GP*AP*GP*GP*U...
+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: MAGNESIUM ION
+Macromolecule #9: ZINC ION
-Experimental details
-Structure determination
Method | ![]() |
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Aggregation state | particle |
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Sample preparation
Concentration | 3 mg/mL | |||||||||||||||
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Buffer | pH: 8 Component:
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Grid | Model: C-flat CF-1.2/1.3-3Au / Material: GOLD / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV | |||||||||||||||
Details | This sample was monodisperse |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Calibrated defocus max: 2.5 µm / Calibrated defocus min: 0.8 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD![]() |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
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: 3-50 / Number grids imaged: 2 / Number real images: 4000 / Average exposure time: 15.0 sec. / Average electron dose: 85.2 e/Å2 |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Particle selection | Number selected: 305200 |
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CTF correction | Software - Name: CTFFIND4 (ver. 4) / Details: CTF parameters were estimated by CTFFIND4 |
Startup model | Type of model: OTHER Details: E.coli core enzyme structure were extracted from PDB 4LLG, and converted to map by using EMAN2 command e2pdb2mrc.py |
Initial angle assignment | Type: OTHER / Software - Name: RELION (ver. 1.4) |
Final 3D classification | Number classes: 6 / Avg.num./class: 50800 / Software - Name: RELION (ver. 2.0) |
Final angle assignment | Type: OTHER / Software - Name: RELION (ver. 1.4) |
Final reconstruction | Number classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.38 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 162400 |
-Atomic model buiding 1
Refinement | Space: REAL |
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Output model | ![]() PDB-6alh: |