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- EMDB-9792: CryoEM structure of Escherichia coli sigmaE transcription initiat... -

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Entry
Database: EMDB / ID: EMD-9792
TitleCryoEM structure of Escherichia coli sigmaE transcription initiation complex containing 5nt of RNA
Map data
SampleEscherichia coli sigmaE transcription initiation complex containing 5nt of RNA
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • ECF RNA polymerase sigma-E factor
  • (nucleic-acidNucleic acid) x 3
  • (ligand) x 2
Function / homology
Function and homology information


response to temperature stimulus / RNA polymerase complex / sigma factor activity / response to osmotic stress / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-templated transcription, initiation / ec:2.7.7.6: / protein-containing complex assembly / protein dimerization activity ...response to temperature stimulus / RNA polymerase complex / sigma factor activity / response to osmotic stress / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-templated transcription, initiation / ec:2.7.7.6: / protein-containing complex assembly / protein dimerization activity / transcription, DNA-templated / DNA-binding transcription factor activity / regulation of transcription, DNA-templated / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm
RNA polymerase sigma factor 70, region 4 type 2 / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / DNA-directed RNA polymerase, alpha subunit / RNA polymerase subunit, RPB6/omega / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase beta subunit / RNA polymerase sigma factor, region 3/4-like / RNA polymerase sigma factor, region 2 ...RNA polymerase sigma factor 70, region 4 type 2 / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / DNA-directed RNA polymerase, alpha subunit / RNA polymerase subunit, RPB6/omega / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase beta subunit / RNA polymerase sigma factor, region 3/4-like / RNA polymerase sigma factor, region 2 / RNA polymerase sigma-70 like domain / RNA polymerase sigma-70 RpoE type / RNA polymerase Rpb2, OB-fold / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, beta subunit, external 1 domain / Winged helix-like DNA-binding domain superfamily / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase, RBP11-like subunit / DNA-directed RNA polymerase, insert domain superfamily / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase sigma-70 like / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb2, domain 6 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb6 / RNA polymerase Rpb3/Rpb11 dimerisation domain / Bacterial RNA polymerase, alpha chain C terminal domain / Sigma-70 region 2 / RNA polymerase, alpha subunit, C-terminal / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, domain 2 / RNA polymerase sigma factor 70, ECF, conserved site / RNA polymerases beta chain signature. / Sigma-70 factors ECF subfamily signature. / RNA polymerase beta subunit external 1 domain / Sigma-70, region 4 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 3 / RNA polymerase, beta subunit, protrusion / RNA polymerase, alpha subunit / RNA polymerase Rpb2, domain 3 / DNA-directed RNA polymerase, omega subunit / RNA polymerase, subunit omega/K/RPB6 / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 7 / RNA polymerase sigma-70 region 2 / RNA polymerase, beta subunit, conserved site / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 3 / RNA polymerase, N-terminal / RNA polymerase Rpb2, domain 7
DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / ECF RNA polymerase sigma-E factor
Biological speciesEscherichia coli K-12 (bacteria) / Escherichia coli (strain K12) (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.02 Å
AuthorsFang CL / Zhang Y
CitationJournal: Nucleic Acids Res. / Year: 2019
Title: Structures and mechanism of transcription initiation by bacterial ECF factors.
Authors: Chengli Fang / Lingting Li / Liqiang Shen / Jing Shi / Sheng Wang / Yu Feng / Yu Zhang /
Abstract: Bacterial RNA polymerase (RNAP) forms distinct holoenzymes with extra-cytoplasmic function (ECF) σ factors to initiate specific gene expression programs. In this study, we report a cryo-EM structure ...Bacterial RNA polymerase (RNAP) forms distinct holoenzymes with extra-cytoplasmic function (ECF) σ factors to initiate specific gene expression programs. In this study, we report a cryo-EM structure at 4.0 Å of Escherichia coli transcription initiation complex comprising σE-the most-studied bacterial ECF σ factor (Ec σE-RPo), and a crystal structure at 3.1 Å of Mycobacterium tuberculosis transcription initiation complex with a chimeric σH/E (Mtb σH/E-RPo). The structure of Ec σE-RPo reveals key interactions essential for assembly of E. coli σE-RNAP holoenzyme and for promoter recognition and unwinding by E. coli σE. Moreover, both structures show that the non-conserved linkers (σ2/σ4 linker) of the two ECF σ factors are inserted into the active-center cleft and exit through the RNA-exit channel. We performed secondary-structure prediction of 27,670 ECF σ factors and find that their non-conserved linkers probably reach into and exit from RNAP active-center cleft in a similar manner. Further biochemical results suggest that such σ2/σ4 linker plays an important role in RPo formation, abortive production and promoter escape during ECF σ factors-mediated transcription initiation.
Validation ReportPDB-ID: 6jbq

SummaryFull reportAbout validation report
DateDeposition: Jan 26, 2019 / Header (metadata) release: May 29, 2019 / Map release: May 29, 2019 / Update: Jun 5, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6jbq
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_9792.map.gz / Format: CCP4 / Size: 35.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.01 Å/pix.
x 210 pix.
= 212.94 Å
1.01 Å/pix.
x 210 pix.
= 212.94 Å
1.01 Å/pix.
x 210 pix.
= 212.94 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.014 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.13787654 - 0.21388943
Average (Standard dev.)0.0012962292 (±0.009455728)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions210210210
Spacing210210210
CellA=B=C: 212.94002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0141.0141.014
M x/y/z210210210
origin x/y/z0.0000.0000.000
length x/y/z212.940212.940212.940
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS210210210
D min/max/mean-0.1380.2140.001

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Supplemental data

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Sample components

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Entire Escherichia coli sigmaE transcription initiation complex containi...

EntireName: Escherichia coli sigmaE transcription initiation complex containing 5nt of RNA
Number of components: 11

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Component #1: protein, Escherichia coli sigmaE transcription initiation complex...

ProteinName: Escherichia coli sigmaE transcription initiation complex containing 5nt of RNA
Recombinant expression: No
MassTheoretical: 443.8 kDa
SourceSpecies: Escherichia coli K-12 (bacteria) / Strain: K-12
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, DNA-directed RNA polymerase subunit alpha

ProteinName: DNA-directed RNA polymerase subunit alphaPolymerase / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 36.55868 kDa
SourceSpecies: Escherichia coli (strain K12) (bacteria) / Strain: K12
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, DNA-directed RNA polymerase subunit beta

ProteinName: DNA-directed RNA polymerase subunit betaPolymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 150.820875 kDa
SourceSpecies: Escherichia coli (strain K12) (bacteria) / Strain: K12
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, DNA-directed RNA polymerase subunit beta'

ProteinName: DNA-directed RNA polymerase subunit beta'Polymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 156.537031 kDa
SourceSpecies: Escherichia coli (strain K12) (bacteria) / Strain: K12
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #5: protein, DNA-directed RNA polymerase subunit omega

ProteinName: DNA-directed RNA polymerase subunit omegaPolymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 10.249547 kDa
SourceSpecies: Escherichia coli (strain K12) (bacteria) / Strain: K12
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: protein, ECF RNA polymerase sigma-E factor

ProteinName: ECF RNA polymerase sigma-E factor / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 25.113385 kDa
SourceSpecies: Escherichia coli (strain K12) (bacteria) / Strain: K12
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #7: nucleic-acid, DNA (48-MER)

nucleic acidName: DNA (48-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DC)(DT)(DG)(DC)(DA)(DT)(DC)(DC)(DG)(DT) (DG)(DA)(DG)(DT)(DC)(DG)(DA)(DG)(DG)(DG) (DT)(DG)(DT)(DT)(DC)(DA)(DA)(DT)(DA)(DA) (DT)(DT)(DA)(DG)(DC)(DA)(DC)(DT)(DA)(DA) (DA)(DA)(DG)(DT)(DT)(DC)(DC)(DG)
MassTheoretical: 14.823522 kDa
SourceSpecies: synthetic construct (others)

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Component #8: nucleic-acid, DNA (48-MER)

nucleic acidName: DNA (48-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DC)(DG)(DG)(DA)(DA)(DC)(DT)(DT)(DT)(DT) (DA)(DG)(DT)(DG)(DC)(DT)(DA)(DA)(DT)(DT) (DA)(DT)(DT)(DG)(DT)(DC)(DA)(DA)(DA)(DA) (DC)(DC)(DA)(DT)(DT)(DG)(DT)(DC)(DA)(DC) (DG)(DG)(DA)(DT)(DG)(DC)(DA)(DG)
MassTheoretical: 14.797521 kDa
SourceSpecies: synthetic construct (others)

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Component #9: nucleic-acid, RNA (5'-R(*CP*UP*CP*GP*A)-3')

nucleic acidName: RNA (5'-R(*CP*UP*CP*GP*A)-3') / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
CUCGA
MassTheoretical: 1.545984 kDa
SourceSpecies: synthetic construct (others)

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Component #10: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Component #11: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 6.540905 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 15 mg/mL / pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 282.65 K / Humidity: 95 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.665 e/Å2 / Illumination mode: FLOOD BEAM
LensCs: 2.7 mm / Imaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 2089

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 195058
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION / Resolution: 4.02 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body
Input PDB model: 4MEY, 2H27, 4LUP
Chain ID: A, A, A
Output model

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