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- EMDB-9916: CryoEM structure of sigma appropriation complex -

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Basic information

Entry
Database: EMDB / ID: EMD-9916
TitleCryoEM structure of sigma appropriation complex
Map data
Samplesigma appropriation complex
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • RNA polymerase sigma factor RpoD
  • 10 kDa anti-sigma factor
  • Middle transcription regulatory protein motA
  • (nucleic-acidNucleic acid) x 2
  • (ligand) x 2
Function / homology
Function and homology information


RNA polymerase complex / transcription initiation from bacterial-type RNA polymerase promoter / sigma factor activity / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein-containing complex assembly / response to heat / protein dimerization activity / transcription, DNA-templated ...RNA polymerase complex / transcription initiation from bacterial-type RNA polymerase promoter / sigma factor activity / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein-containing complex assembly / response to heat / protein dimerization activity / transcription, DNA-templated / response to antibiotic / regulation of transcription, DNA-templated / DNA-binding transcription factor activity / DNA binding / zinc ion binding / membrane / metal ion binding / cytosol / cytoplasm
Anti-Sigma Factor A / Bacteriophage T4, MotA, transcription regulator N-terminal / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / RPB6/omega subunit-like superfamily / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase, beta subunit, external 1 domain / DNA-directed RNA polymerase, subunit 2 / Transcription regulator MotA, C-terminal / RNA polymerase Rpb2, OB-fold ...Anti-Sigma Factor A / Bacteriophage T4, MotA, transcription regulator N-terminal / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / RPB6/omega subunit-like superfamily / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase, beta subunit, external 1 domain / DNA-directed RNA polymerase, subunit 2 / Transcription regulator MotA, C-terminal / RNA polymerase Rpb2, OB-fold / Anti-Sigma Factor A superfamily / RNA polymerase sigma-70 like domain / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / RNA polymerase sigma factor RpoD, C-terminal / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase subunit, RPB6/omega / DNA-directed RNA polymerase, alpha subunit / Transcription regulator MotA, C-terminal domain superfamily / RNA polymerase, RBP11-like subunit / DNA-directed RNA polymerase, insert domain / RNA polymerase Rpb6 / Sigma-70, non-essential region / Sigma-70, region 4 / Sigma-70 region 2 / Sigma-70 region 3 / Sigma-70 factor, region 1.1 / Bacterial RNA polymerase, alpha chain C terminal domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase Rpb3/RpoA insert domain / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb2, domain 6 / Sigma-70 factor, region 1.2 / Sigma-70 factor, region 1.1 superfamily / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase, alpha subunit, C-terminal / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase sigma-70 / RNA polymerase, alpha subunit / RNA polymerase beta subunit / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 4 / RNA polymerase, subunit omega/K/RPB6 / Anti-Sigma Factor A / Transcription factor MotA, activation domain / Bacteriophage T4 MotA, C-terminal / RNA polymerase beta subunit external 1 domain / Sigma-70 factors family signature 1. / Sigma-70 factors family signature 2. / RNA polymerases beta chain signature. / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, omega subunit / RNA polymerase, N-terminal / RNA polymerase sigma-70 region 3 / RNA polymerase sigma-70 region 1.2 / RNA polymerase Rpb2, domain 3 / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 7 / RNA polymerase sigma factor 70, non-essential domain / RNA polymerase sigma-70 region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb1, domain 3 / RNA polymerase sigma factor 70, region 1.1 / 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 sigma factor RpoD / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / Middle transcription regulatory protein motA / 10 kDa anti-sigma factor
Biological speciesEscherichia coli K-12 (bacteria) / Enterobacteria phage T4 (bacteriophage)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.79 Å
AuthorsShi J / Wen A / Feng Y
Funding support China, 1 items
OrganizationGrant numberCountry
Ministry of Science and Technology (China)2018YFA0507800 China
CitationJournal: Nucleic Acids Res. / Year: 2019
Title: Structural basis of σ appropriation.
Authors: Jing Shi / Aijia Wen / Minxing Zhao / Linlin You / Yu Zhang / Yu Feng /
Abstract: Bacteriophage T4 middle promoters are activated through a process called σ appropriation, which requires the concerted effort of two T4-encoded transcription factors: AsiA and MotA. Despite ...Bacteriophage T4 middle promoters are activated through a process called σ appropriation, which requires the concerted effort of two T4-encoded transcription factors: AsiA and MotA. Despite extensive biochemical and genetic analyses, puzzle remains, in part, because of a lack of precise structural information for σ appropriation complex. Here, we report a single-particle cryo-electron microscopy (cryo-EM) structure of an intact σ appropriation complex, comprising AsiA, MotA, Escherichia coli RNA polymerase (RNAP), σ70 and a T4 middle promoter. As expected, AsiA binds to and remodels σ region 4 to prevent its contact with host promoters. Unexpectedly, AsiA undergoes a large conformational change, takes over the job of σ region 4 and provides an anchor point for the upstream double-stranded DNA. Because σ region 4 is conserved among bacteria, other transcription factors may use the same strategy to alter the landscape of transcription immediately. Together, the structure provides a foundation for understanding σ appropriation and transcription activation.
Validation ReportPDB-ID: 6k4y

SummaryFull reportAbout validation report
History
DepositionMay 27, 2019-
Header (metadata) releaseJul 31, 2019-
Map releaseAug 7, 2019-
UpdateAug 7, 2019-
Current statusAug 7, 2019Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0278
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0278
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6k4y
  • Surface level: 0.0278
  • 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_9916.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 200 pix.
= 261.4 Å
1.31 Å/pix.
x 200 pix.
= 261.4 Å
1.31 Å/pix.
x 200 pix.
= 261.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.307 Å
Density
Contour LevelBy AUTHOR: 0.0278 / Movie #1: 0.0278
Minimum - Maximum-0.14411527 - 0.21478961
Average (Standard dev.)0.00009131287 (±0.007976222)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 261.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3071.3071.307
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z261.400261.400261.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ344344344
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.1440.2150.000

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Supplemental data

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Sample components

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Entire sigma appropriation complex

EntireName: sigma appropriation complex / Number of components: 12

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Component #1: protein, sigma appropriation complex

ProteinName: sigma appropriation complex / Recombinant expression: No
SourceSpecies: Escherichia coli K-12 (bacteria)

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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 K-12 (bacteria) / Strain: K-12
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 K-12 (bacteria) / Strain: K-12
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: 155.366781 kDa
SourceSpecies: Escherichia coli K-12 (bacteria) / Strain: K-12
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 K-12 (bacteria) / Strain: K-12
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: protein, RNA polymerase sigma factor RpoD

ProteinName: RNA polymerase sigma factor RpoD / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 70.352242 kDa
SourceSpecies: Escherichia coli K-12 (bacteria) / Strain: K-12
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #7: protein, 10 kDa anti-sigma factor

ProteinName: 10 kDa anti-sigma factor / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 12.775375 kDa
SourceSpecies: Enterobacteria phage T4 (bacteriophage)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #8: protein, Middle transcription regulatory protein motA

ProteinName: Middle transcription regulatory protein motA / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 23.608334 kDa
SourceSpecies: Enterobacteria phage T4 (bacteriophage)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #9: nucleic-acid, DNA (60-MER)

nucleic acidName: DNA (60-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DC)(DG)(DA)(DA)(DA)(DA)(DG)(DA)(DA)(DG) (DC)(DT)(DT)(DT)(DG)(DC)(DT)(DT)(DA)(DA) (DT)(DA)(DA)(DT)(DC)(DC)(DA)(DT)(DA)(DT) (DG)(DG)(DT)(DT)(DA)(DT)(DA)(DA)(DT)(DG) (DG)(DG)(DA)(DG)(DC)(DT)(DG)(DT)(DC)(DA) (DC)(DG)(DG)(DA)(DT)(DG)(DC)(DA)(DG)(DG)
MassTheoretical: 18.642982 kDa
SourceSpecies: Enterobacteria phage T4 (bacteriophage)

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Component #10: nucleic-acid, DNA (60-MER)

nucleic acidName: DNA (60-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DC)(DC)(DT)(DG)(DC)(DA)(DT)(DC)(DC)(DG) (DT)(DG)(DA)(DG)(DT)(DC)(DG)(DA)(DG)(DG) (DG)(DT)(DA)(DA)(DT)(DA)(DA)(DA)(DC)(DC) (DA)(DT)(DA)(DT)(DG)(DG)(DA)(DT)(DT)(DA) (DT)(DT)(DA)(DA)(DG)(DC)(DA)(DA)(DA)(DG) (DC)(DT)(DT)(DC)(DT)(DT)(DT)(DT)(DC)(DG)
MassTheoretical: 18.504877 kDa
SourceSpecies: Enterobacteria phage T4 (bacteriophage)

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Component #11: ligand, MAGNESIUM ION

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

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Component #12: 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 solutionpH: 7.9
VitrificationCryogen name: ETHANE

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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: 56 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 105108
3D reconstructionResolution: 3.79 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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