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- EMDB-0700: cryo-EM structure of Escherichia coli Crl transcription activatio... -

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Basic information

Entry
Database: EMDB / ID: EMD-0700
Titlecryo-EM structure of Escherichia coli Crl transcription activation complex
Map data
SampleEscherichia coli Crl transcription activation complex(Crl-TAC)
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • RNA polymerase sigma factor RpoS
  • (nucleic-acidNucleic acid) x 3
  • Sigma factor-binding protein Crl
  • (ligand) x 2
Function / homology
Function and homology information


bacterial-type RNA polymerase core enzyme binding / RNA polymerase complex / DNA-templated transcription, initiation => GO:0006352 / sigma factor activity / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein-containing complex assembly / transcription, DNA-templated / protein dimerization activity ...bacterial-type RNA polymerase core enzyme binding / RNA polymerase complex / DNA-templated transcription, initiation => GO:0006352 / sigma factor activity / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein-containing complex assembly / transcription, DNA-templated / protein dimerization activity / response to antibiotic / positive regulation of transcription, DNA-templated / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
Sigma factor-binding transcriptional regulator Crl / Sigma factor-binding transcriptional regulator Crl / Sigma factor-binding protein Crl superfamily / RNA polymerase sigma factor RpoS / Sigma-70 factors family signature 1. / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / Sigma-70 factors family signature 2. / Sigma-70 region 3 / RNA polymerase sigma-70 region 3 ...Sigma factor-binding transcriptional regulator Crl / Sigma factor-binding transcriptional regulator Crl / Sigma factor-binding protein Crl superfamily / RNA polymerase sigma factor RpoS / Sigma-70 factors family signature 1. / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / Sigma-70 factors family signature 2. / Sigma-70 region 3 / RNA polymerase sigma-70 region 3 / RNA polymerase sigma-70 / Sigma-70, region 4 / RNA polymerase sigma-70 region 4 / RNA polymerase sigma-70 like domain / Sigma-70 region 2 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / Bacterial RNA polymerase, alpha chain C terminal domain / RNA polymerase, alpha subunit, C-terminal / RNA polymerase beta subunit external 1 domain / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, beta subunit, external 1 domain / DNA-directed RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb6 / RNA polymerase Rpb6 / RPB6/omega subunit-like superfamily / RNA polymerase, subunit omega/Rpo6/RPB6 / : / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb1, domain 2 / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase beta subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase Rpb3/RpoA insert domain / RNA polymerases D / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, domain 3 / RNA polymerase, beta subunit, conserved site / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / RNA polymerase Rpb2, domain 6 / RNA polymerase Rpb2, domain 7 / DNA-directed RNA polymerase, subunit 2 / RNA polymerase Rpb2, OB-fold / RNA polymerase Rpb2, domain 7 / RNA polymerases beta chain signature. / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
Sigma factor-binding protein Crl / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / RNA polymerase sigma factor RpoS
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria) / Escherichia coli (strain K12) (bacteria) / synthetic construct (others) / Escherichia coli (strain ATCC 8739 / DSM 1576 / Crooks) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsXu J / Zhang Y
Funding support China, 2 items
OrganizationGrant numberCountry
National Natural Science Foundation of China31822001 China
Chinese Academy of SciencesXDB29020000 China
CitationJournal: Elife / Year: 2019
Title: Crl activates transcription by stabilizing active conformation of the master stress transcription initiation factor.
Authors: Juncao Xu / Kaijie Cui / Liqiang Shen / Jing Shi / Lingting Li / Linlin You / Chengli Fang / Guoping Zhao / Yu Feng / Bei Yang / Yu Zhang /
Abstract: σ is a master transcription initiation factor that protects bacterial cells from various harmful environmental stresses including antibiotic pressure. Although its mechanism remains unclear, it is ...σ is a master transcription initiation factor that protects bacterial cells from various harmful environmental stresses including antibiotic pressure. Although its mechanism remains unclear, it is known that full activation of σ-mediated transcription requires a σ-specific activator, Crl. In this study, we determined a 3.80 Å cryo-EM structure of an transcription activation complex ( Crl-TAC) comprising σ-RNA polymerase (σ-RNAP) holoenzyme, Crl, and a nucleic-acid scaffold. The structure reveals that Crl interacts with domain 2 of σ (σ) and the RNAP core enzyme, but does not contact promoter DNA. Results from subsequent hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicate that Crl stabilizes key structural motifs within σ to promote the assembly of the σ-RNAP holoenzyme and also to facilitate formation of an RNA polymerase-promoter DNA open complex (RPo). Our study demonstrates a unique DNA contact-independent mechanism of transcription activation, thereby defining a previously unrecognized mode of transcription activation in cells.
History
DepositionJul 21, 2019-
Header (metadata) releaseJan 1, 2020-
Map releaseJan 1, 2020-
UpdateDec 9, 2020-
Current statusDec 9, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0156
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0156
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6kj6
  • Surface level: 0.0156
  • 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_0700.map.gz / Format: CCP4 / Size: 52.7 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 240 pix.
= 243.36 Å
1.01 Å/pix.
x 240 pix.
= 243.36 Å
1.01 Å/pix.
x 240 pix.
= 243.36 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: 0.0156 / Movie #1: 0.0156
Minimum - Maximum-0.06503575 - 0.10948706
Average (Standard dev.)0.00039438438 (±0.0035550676)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 243.36002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0141.0141.014
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z243.360243.360243.360
α/β/γ90.00090.00090.000
start NX/NY/NZ-31-35-52
NX/NY/NZ11798209
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0650.1090.000

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Supplemental data

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Half map: #2

Fileemd_0700_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_0700_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Escherichia coli Crl transcription activation complex(Crl-TAC)

EntireName: Escherichia coli Crl transcription activation complex(Crl-TAC)
Number of Components: 12

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Component #1: protein, Escherichia coli Crl transcription activation complex(Cr...

ProteinName: Escherichia coli Crl transcription activation complex(Crl-TAC)
Recombinant expression: No
MassTheoretical: 440 kDa
SourceSpecies: Escherichia coli K-12 (bacteria) / Strain: K-12
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)

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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 BL21(DE3) (bacteria)

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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 BL21(DE3) (bacteria)

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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 BL21(DE3) (bacteria)

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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 BL21(DE3) (bacteria)

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Component #6: protein, RNA polymerase sigma factor RpoS

ProteinName: RNA polymerase sigma factor RpoS / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 38.352211 kDa
SourceSpecies: Escherichia coli (strain K12) (bacteria) / Strain: K12
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #7: nucleic-acid, DNA (51-MER)

nucleic acidName: DNA (51-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DC)(DC)(DT)(DG)(DC)(DA)(DT)(DC)(DC)(DG) (DT)(DG)(DC)(DG)(DT)(DC)(DG)(DA)(DG)(DG) (DG)(DT)(DA)(DA)(DT)(DA)(DA)(DG)(DC)(DA) (DT)(DA)(DC)(DG)(DT)(DG)(DA)(DG)(DG)(DT) (DC)(DC)(DG)(DA)(DT)(DG)(DT)(DC)(DA)(DA) (DG)
MassTheoretical: 15.797117 kDa
SourceSpecies: synthetic construct (others)

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Component #8: nucleic-acid, DNA (51-MER)

nucleic acidName: DNA (51-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DC)(DT)(DT)(DG)(DA)(DC)(DA)(DT)(DC)(DG) (DG)(DA)(DC)(DC)(DT)(DC)(DA)(DC)(DG)(DT) (DA)(DT)(DG)(DC)(DT)(DA)(DT)(DA)(DA)(DT) (DG)(DG)(DG)(DA)(DG)(DC)(DT)(DG)(DG)(DC) (DA)(DC)(DG)(DG)(DA)(DT)(DG)(DC)(DA)(DG) (DG)
MassTheoretical: 15.797115 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: protein, Sigma factor-binding protein Crl

ProteinName: Sigma factor-binding protein Crl / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 16.009337 kDa
SourceSpecies: Escherichia coli (strain ATCC 8739 / DSM 1576 / Crooks) (bacteria)
Strain: ATCC 8739 / DSM 1576 / Crooks
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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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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Component #12: ligand, MAGNESIUM ION

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

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Experimental details

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Sample preparation

SpecimenSpecimen State: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 12 mg/mL / pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen Name: ETHANE / Temperature: 283 K / Humidity: 95 % / Details: blot for 8 seconds before plunging.

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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: 1.675 e/Å2 / Illumination Mode: FLOOD BEAM
LensCs: 2.7 mm / Imaging Mode: BRIGHT FIELD / Defocus: 2000.0 - 2600.0 nm
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of Digital Images: 3290

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Image processing

ProcessingMethod: single particle reconstruction / Applied Symmetry: C1 (asymmetric) / Number of Projections: 184208
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION / Resolution: 3.8 Å / Resolution Method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 5IPL, 4Q11
Output model

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