- EMDB-9970: Thermococcus kodakarensis RNA polymerase in complex with transcri... -
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Open data
ID or keywords:
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Basic information
Entry
Database: EMDB / ID: EMD-9970
Title
Thermococcus kodakarensis RNA polymerase in complex with transcription factor E and promoter DNA, focused refinement on RNAP stalk and clamp domains and TFE
Map data
Cryo-EM single particle analysis 3D reconstruction of RNA polymerase (RNAP) from Thermococcus kodakarensis (Tko) in complex with transcription factor E (TFE) and DNA, focused refinement
Sample
Complex: Thermococcus kodakarensis RNA polymerase in complex with transcription factor E and promoter DNA
Function / homology
Function and homology information
transcription open complex formation at RNA polymerase II promoter / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / nucleotide binding / DNA-templated transcription / regulation of DNA-templated transcription ...transcription open complex formation at RNA polymerase II promoter / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / nucleotide binding / DNA-templated transcription / regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm Similarity search - Function
Download / File: emd_9970.map.gz / Format: CCP4 / Size: 20.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
Cryo-EM single particle analysis 3D reconstruction of RNA polymerase (RNAP) from Thermococcus kodakarensis (Tko) in complex with transcription factor E (TFE) and DNA, focused refinement
Entire : Thermococcus kodakarensis RNA polymerase in complex with transcri...
Entire
Name: Thermococcus kodakarensis RNA polymerase in complex with transcription factor E and promoter DNA
Components
Complex: Thermococcus kodakarensis RNA polymerase in complex with transcription factor E and promoter DNA
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Supramolecule #1: Thermococcus kodakarensis RNA polymerase in complex with transcri...
Supramolecule
Name: Thermococcus kodakarensis RNA polymerase in complex with transcription factor E and promoter DNA type: complex / ID: 1 / Parent: 0 Details: RNA polymerase from euryarchaea (Thermococcus kodakarensis) composed of 11 subunits (Rpo1', Rpo1", Rpo2, Rpo3, Rpo4, Rpo5, Rpo6, Rpo7, Rpo10, Rpo11, Rpo12), and in complex with transcription ...Details: RNA polymerase from euryarchaea (Thermococcus kodakarensis) composed of 11 subunits (Rpo1', Rpo1", Rpo2, Rpo3, Rpo4, Rpo5, Rpo6, Rpo7, Rpo10, Rpo11, Rpo12), and in complex with transcription factor E and promoter DNA
Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #0 - topology: HOLEY / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: GRAPHENE OXIDE / Support film - #1 - topology: HOLEY
Vitrification
Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV
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Electron microscopy
Microscope
FEI TITAN KRIOS
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Film or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Frames/image: 1-30 / Number real images: 3754 / Average exposure time: 1.8 sec. / Average electron dose: 63.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
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Image processing
Particle selection
Number selected: 1388666 Details: Approximately 10,000 particles were selected manually. 2D class averages from the manual-picked particles were used as a template for automated particle picking in Relion 2.1.
CTF correction
Software - Name: Gctf (ver. 1.06) Details: Defocus parameters were calculated from motion-corrected micrographs, using Gctf 1.06. Using the defocus parameters, Cs (0.003, after Cs image corrector tuning) and amplitude contrast (0.1), ...Details: Defocus parameters were calculated from motion-corrected micrographs, using Gctf 1.06. Using the defocus parameters, Cs (0.003, after Cs image corrector tuning) and amplitude contrast (0.1), CTF phase flipping and amplitude correction was performed during the reconstruction using Relion 2.1.
Details: PDB file was converted to EM map using e2pdb2mrc.py routine in EMAN2 software package. The reference map was lowpass filtered to 60 angstroms as a startup model.
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final reconstruction
Number classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.85 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) Details: Focused refinement on RNAP stalk and clamp domains and TFE was performed by continuing the last iteration of the 3D refinement run of the entire structure (EMD-9962), with the applied local mask Number images used: 312092
Details
Movie frame data was subjected to motion correction to generate summed micrographs using MotionCor2.
FSC plot (resolution estimation)
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