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- EMDB-9969: Thermococcus kodakarensis RNA polymerase in complex with transcri... -

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Basic information

Entry
Database: EMDB / ID: EMD-9969
TitleThermococcus kodakarensis RNA polymerase in complex with transcription factor E, focused refinement on RNAP stalk and clamp domains and TFE
Map dataCryo-EM single particle analysis 3D reconstruction of RNA polymerase (RNAP) from Thermococcus kodakarensis (Tko) in complex with transcription factor E (TFE), resulting from focused refinement
Sample
  • Complex: Thermococcus kodakarensis RNA polymerase in complex with transcription factor E
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 / chromosome / protein dimerization activity / nucleotide binding / 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 / chromosome / 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
Transcription factor TFE, archaea / DNA-directed RNA polymerase subunit Rpo4 / DNA-directed RNA polymerase subunit Rpo12 / DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit Rpo2 / DNA-directed RNA polymerase subunit Rpo7 / HRDC domain superfamily / DNA-directed RNA polymerase, subunit E/RPC8 / Transcription initiation factor IIE subunit alpha, N-terminal ...Transcription factor TFE, archaea / DNA-directed RNA polymerase subunit Rpo4 / DNA-directed RNA polymerase subunit Rpo12 / DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit Rpo2 / DNA-directed RNA polymerase subunit Rpo7 / HRDC domain superfamily / DNA-directed RNA polymerase, subunit E/RPC8 / Transcription initiation factor IIE subunit alpha, N-terminal / Transcription factor TFE/TFIIEalpha HTH domain / TFIIEalpha/SarR/Rpc3 HTH domain / Transcription factor E / TFIIE alpha subunit / TFE/IIEalpha-type HTH domain profile. / Transcription initiation factor IIE / RNA polymerase subunit Rpb4/RPC9 / RNA polymerase Rpb4 / S1 domain profile. / HRDC-like superfamily / RNA polymerase Rpb7-like , N-terminal / RNA polymerase Rpb7-like, N-terminal domain superfamily / RNA polymerase subunit Rpb7-like / SHS2 domain found in N terminus of Rpb7p/Rpc25p/MJ0397 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 5 / DNA-directed RNA polymerase subunit RPABC5/Rpb10 / RNA polymerases, subunit N, zinc binding site / RNA polymerase subunit RPB10 / RNA polymerases N / 8 kDa subunit / RNA polymerases N / 8 Kd subunits signature. / : / DNA directed RNA polymerase, 7 kDa subunit / RNA polymerase archaeal subunit P/eukaryotic subunit RPABC4 / RNA polymerase, subunit H/Rpb5, conserved site / RNA polymerases H / 23 Kd subunits signature. / RNA polymerase subunit CX / DNA-directed RNA polymerase, 30-40kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo3/Rpb3/RPAC1 / RNA polymerases D / 30 to 40 Kd subunits signature. / DNA-directed RNA polymerase Rpb11, 13-16kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo11 / RNA polymerases L / 13 to 16 Kd subunits signature. / DNA-directed RNA polymerase subunit Rpo5/Rpb5 / DNA-directed RNA polymerase, RBP11-like dimerisation domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase, subunit H/Rpb5 C-terminal / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / RPB5-like RNA polymerase subunit superfamily / RNA polymerase Rpb5, C-terminal domain / Archaeal Rpo6/eukaryotic RPB6 RNA polymerase subunit / DNA-directed RNA polymerase, 14-18kDa subunit, conserved site / RNA polymerases K / 14 to 18 Kd subunits signature. / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, OB-fold / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerases beta chain signature. / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 6
Similarity search - Domain/homology
Transcription factor E / DNA-directed RNA polymerase subunit Rpo12 / DNA-directed RNA polymerase subunit Rpo5 / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit Rpo11 / DNA-directed RNA polymerase subunit Rpo4 / DNA-directed RNA polymerase subunit Rpo7 / DNA-directed RNA polymerase subunit Rpo10 ...Transcription factor E / DNA-directed RNA polymerase subunit Rpo12 / DNA-directed RNA polymerase subunit Rpo5 / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit Rpo11 / DNA-directed RNA polymerase subunit Rpo4 / DNA-directed RNA polymerase subunit Rpo7 / DNA-directed RNA polymerase subunit Rpo10 / DNA-directed RNA polymerase subunit Rpo6 / DNA-directed RNA polymerase subunit Rpo3
Similarity search - Component
Biological speciesThermococcus kodakarensis KOD1 (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.25 Å
AuthorsHyun J / Jun S-H / Cho H-S / Murakami KS
Funding support United States, Korea, Republic Of, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research InstituteR35 GM131860 United States
Ministry of Education (Korea)NRF-2017M3A9F6029755 Korea, Republic Of
Ministry of Education (Korea)2015R1D1A1A01059097 Korea, Republic Of
Ministry of Education (Korea)NRF-2019M3E5D6063903 Korea, Republic Of
CitationJournal: To Be Published
Title: Thermococcus kodakraensis RNA polymerase in apo form
Authors: Hyun J / Jun S-H / Cho H-S / Murakami KS
History
DepositionJul 6, 2019-
Header (metadata) releaseJul 1, 2020-
Map releaseJul 1, 2020-
UpdateDec 23, 2020-
Current statusDec 23, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6kf4
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9969.png

Downloads & links

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Map

FileDownload / File: emd_9969.map.gz / Format: CCP4 / Size: 20.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM single particle analysis 3D reconstruction of RNA polymerase (RNAP) from Thermococcus kodakarensis (Tko) in complex with transcription factor E (TFE), resulting from focused refinement
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.4 Å/pix.
x 176 pix.
= 246.4 Å		1.4 Å/pix.
x 176 pix.
= 246.4 Å		1.4 Å/pix.
x 176 pix.
= 246.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.4 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.3365371 - 0.57291895
Average (Standard dev.)0.0018314726 (±0.013435258)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions176176176
Spacing176176176
CellA=B=C: 246.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.41.41.4
M x/y/z176176176
origin x/y/z0.0000.0000.000
length x/y/z246.400246.400246.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS176176176
D min/max/mean-0.3370.5730.002

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Supplemental data

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Mask #1

Fileemd_9969_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Tko RNAP-TFE (focused refinement) half map 2

Fileemd_9969_half_map_1.map
AnnotationTko RNAP-TFE (focused refinement) half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Tko RNAP-TFE (focused refinement) half map 1

Fileemd_9969_half_map_2.map
AnnotationTko RNAP-TFE (focused refinement) half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Thermococcus kodakarensis RNA polymerase in complex with transcri...

EntireName: Thermococcus kodakarensis RNA polymerase in complex with transcription factor E
Components
  • Complex: Thermococcus kodakarensis RNA polymerase in complex with transcription factor E

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Supramolecule #1: Thermococcus kodakarensis RNA polymerase in complex with transcri...

SupramoleculeName: Thermococcus kodakarensis RNA polymerase in complex with transcription factor E
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 factor E
Source (natural)Organism: Thermococcus kodakarensis KOD1 (archaea)
Molecular weightTheoretical: 410 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsSpherical aberration corrector: Image Cs corrector
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Frames/image: 1-30 / Number real images: 2698 / Average exposure time: 1.8 sec. / Average electron dose: 63.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 100000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.002 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 47000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsMovie frame data was subjected to motion correction to generate summed micrographs using MotionCor2.
Particle selectionNumber selected: 1226339
Details: Approximately 20,000 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 correctionSoftware - 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.
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4qiw


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.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.25 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1)
Details: Focused refinement on stalk and clamp domains of RNAP and TFE was performed by continuing the last iteration of the 3D refinement run of the entire structure (EMD-9961), with the applied local mask
Number images used: 252508
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 8 / Avg.num./class: 51207 / Software - Name: RELION (ver. 2.1)
FSC plot (resolution estimation)

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