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- EMDB-7059: The cryo-EM structure of a bacterial class I transcription activa... -

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Entry
Database: EMDB / ID: 7059
TitleThe cryo-EM structure of a bacterial class I transcription activation complex
Map dataClass I transcription activation complex
SampleThe complex of class-I bacterial transcription activation complex
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 5
  • RNA polymerase sigma factor RpoD
  • cAMP-activated global transcriptional regulator CRP
  • (nucleic-acidNucleic acid) x 3
  • (ligand) x 3
Function / homologyTranscription regulator HTH, Crp-type, conserved site / Winged helix-like DNA-binding domain superfamily / Cyclic nucleotide-binding domain / 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, insert domain superfamily / RNA polymerase, RBP11-like subunit / Winged helix DNA-binding domain superfamily / RPB6/omega subunit-like superfamily ...Transcription regulator HTH, Crp-type, conserved site / Winged helix-like DNA-binding domain superfamily / Cyclic nucleotide-binding domain / 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, insert domain superfamily / RNA polymerase, RBP11-like subunit / Winged helix DNA-binding domain superfamily / RPB6/omega subunit-like superfamily / RNA polymerase Rpb2, domain 6 / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase, beta subunit, external 1 domain / Cyclic nucleotide-binding-like / Cyclic nucleotide-binding, conserved site / DNA-directed RNA polymerase, subunit 2 / RNA polymerase Rpb2, OB-fold / RmlC-like jelly roll fold / RNA polymerase sigma-70 like domain / Sigma-70 factor, region 1.2 / RNA polymerase Rpb1, domain 2 / Sigma-70 factors family signature 1. / RNA polymerase Rpb2, domain 2 / Crp-like helix-turn-helix domain / RNA polymerase beta subunit external 1 domain / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 3 / RNA polymerase beta subunit / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb3/RpoA insert domain / 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 Rpb6 / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma-70 / 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, subunit omega/K/RPB6 / DNA-directed RNA polymerase, omega subunit / RNA polymerase, alpha subunit / RNA polymerase, beta subunit, conserved site / Cyclic nucleotide-binding domain / Sigma-70 factors family signature 2. / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding domain signature 2. / RNA polymerases beta chain signature. / cAMP/cGMP binding motif profile. / Crp-type HTH domain profile. / DNA-directed RNA polymerase, subunit beta-prime / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / RNA polymerase sigma factor 70, region 1.1 / DNA-directed RNA polymerase beta subunit, bacterial-type / Crp-type HTH domain / RNA polymerase subunit, RPB6/omega / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / DNA-directed RNA polymerase, insert domain / RNA polymerase sigma-70 region 3 / RNA polymerase, alpha subunit, C-terminal / 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 / Crp-type HTH domain signature. / transcription initiation from bacterial-type RNA polymerase promoter / sigma factor activity / cAMP binding / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / protein dimerization activity / transcription, DNA-templated / DNA-binding transcription factor activity / DNA binding / cytosol / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta' / cAMP-activated global transcriptional regulator CRP
Function and homology information
SourceEscherichia coli (E. coli) / Escherichia coli O157:H7 (bacteria) / Escherichia coli O45:K1 (strain S88 / ExPEC) (bacteria) / Escherichia coli O45:K1 (bacteria) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / 3.9 Å resolution
AuthorsLiu B / Hong C / Huang R / Yu Z / Steitz TA
CitationJournal: Science / Year: 2017
Title: Structural basis of bacterial transcription activation.
Authors: Bin Liu / Chuan Hong / Rick K Huang / Zhiheng Yu / Thomas A Steitz
Abstract: In bacteria, the activation of gene transcription at many promoters is simple and only involves a single activator. The cyclic adenosine 3',5'-monophosphate receptor protein (CAP), a classic ...In bacteria, the activation of gene transcription at many promoters is simple and only involves a single activator. The cyclic adenosine 3',5'-monophosphate receptor protein (CAP), a classic activator, is able to activate transcription independently through two different mechanisms. Understanding the class I mechanism requires an intact transcription activation complex (TAC) structure at a high resolution. Here we report a high-resolution cryo-electron microscopy structure of an intact class I TAC containing a CAP dimer, a σ-RNA polymerase (RNAP) holoenzyme, a complete class I CAP-dependent promoter DNA, and a de novo synthesized RNA oligonucleotide. The structure shows how CAP wraps the upstream DNA and how the interactions recruit RNAP. Our study provides a structural basis for understanding how activators activate transcription through the class I recruitment mechanism.
Validation ReportPDB-ID: 6b6h

SummaryFull reportAbout validation report
DateDeposition: Oct 2, 2017 / Header (metadata) release: Nov 1, 2017 / Map release: Nov 15, 2017 / Last update: Jul 18, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.13
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.13
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6b6h
  • Surface level: 0.13
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_7059.map.gz (map file in CCP4 format, 32001 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
200 pix
1.35 Å/pix.
= 270. Å
200 pix
1.35 Å/pix.
= 270. Å
200 pix
1.35 Å/pix.
= 270. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density
Contour Level:0.13 (by author), 0.13 (movie #1):
Minimum - Maximum-0.079181686 - 0.36147064
Average (Standard dev.)0.004876344 (0.022157894)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions200200200
Origin0.0.0.
Limit199.199.199.
Spacing200200200
CellA=B=C: 270.0 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z270.000270.000270.000
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0790.3610.005

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Supplemental data

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Sample components

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Entire The complex of class-I bacterial transcription activation complex

EntireName: The complex of class-I bacterial transcription activation complex
Details: A CAP dimer, a sigma70-RNA polymerase holoenzyme, an intact CAP-dependent promoter DNA, and a de novo synthesized RNA oligonucleotide
Number of components: 14

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Component #1: cellular-component, The complex of class-I bacterial transcriptio...

Cellular-componentName: The complex of class-I bacterial transcription activation complex
Details: A CAP dimer, a sigma70-RNA polymerase holoenzyme, an intact CAP-dependent promoter DNA, and a de novo synthesized RNA oligonucleotide
Recombinant expression: No
MassTheoretical: 590 kDa
SourceSpecies: 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 O157:H7 (bacteria)
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 O45:K1 (strain S88 / ExPEC) (bacteria)
Strain: S88 / ExPEC
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 O157:H7 (bacteria)
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 O45:K1 (bacteria) / Strain: S88 / ExPEC
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: 72.206266 kDa
SourceSpecies: Escherichia coli (strain K12) (bacteria) / Strain: K12
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #7: protein, cAMP-activated global transcriptional regulator CRP

ProteinName: cAMP-activated global transcriptional regulator CRP / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 23.672439 kDa
SourceSpecies: Escherichia coli O157:H7 (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #8: protein, DNA-directed RNA polymerase subunit alpha

ProteinName: DNA-directed RNA polymerase subunit alphaPolymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 8.346699 kDa
SourceSpecies: Escherichia coli O157:H7 (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #9: nucleic-acid, SYNTHETIC NONTEMPLATE STRAND DNA (88-MER)

Nucleic-acidName: SYNTHETIC NONTEMPLATE STRAND DNA (88-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DT)(DA)(DA)(DA)(DA)(DT)(DG)(DT)(DG)(DA) (DT)(DC)(DT)(DA)(DG)(DA)(DT)(DC)(DA)(DC) (DA)(DT)(DT)(DT)(DT)(DA)(DG)(DG)(DC)(DA) (DA)(DA)(DA)(DA)(DA)(DG)(DG)(DC)(DC)(DT) (DT)(DG)(DA)(DC)(DA)(DT)(DC)(DC)(DC)(DA) (DC)(DC)(DT)(DC)(DA)(DC)(DG)(DT)(DA)(DT) (DG)(DC)(DT)(DA)(DT)(DA)(DA)(DT)(DG)(DT) (DG)(DT)(DG)(DC)(DA)(DG)(DT)(DC)(DT)(DG) (DA)(DC)(DG)(DC)(DG)(DG)(DC)(DG)
MassTheoretical: 27.133393 kDa
SourceSpecies: Escherichia coli (E. coli)

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Component #10: nucleic-acid, SYNTHETIC TEMPLATE STRAND DNA (88-MER)

Nucleic-acidName: SYNTHETIC TEMPLATE STRAND DNA (88-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DC)(DG)(DC)(DC)(DG)(DC)(DG)(DT)(DC)(DA) (DG)(DA)(DC)(DT)(DC)(DG)(DT)(DA)(DG)(DG) (DA)(DT)(DT)(DA)(DT)(DA)(DG)(DC)(DA)(DT) (DA)(DC)(DG)(DT)(DG)(DA)(DG)(DG)(DT)(DG) (DG)(DG)(DA)(DT)(DG)(DT)(DC)(DA)(DA)(DG) (DG)(DC)(DC)(DT)(DT)(DT)(DT)(DT)(DT)(DG) (DC)(DC)(DT)(DA)(DA)(DA)(DA)(DT)(DG)(DT) (DG)(DA)(DT)(DC)(DT)(DA)(DG)(DA)(DT)(DC) (DA)(DC)(DA)(DT)(DT)(DT)(DT)(DA)
MassTheoretical: 27.217404 kDa
SourceSpecies: Escherichia coli (E. coli)

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Component #11: nucleic-acid, NASCENT RNA 3-MER

Nucleic-acidName: NASCENT RNA 3-MER / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
(GTP)AG
MassTheoretical: 1.134619 kDa
SourceSpecies: Escherichia coli (E. coli)

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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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Component #13: ligand, MAGNESIUM ION

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

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Component #14: ligand, ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE

LigandName: ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 0.329206 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 3 mg/ml
Buffer solution: 20 mM TRIS pH 7.5, 50 mM sodium chloride, 0.1mM EDTA, 5 mM MgCl2, 5 mM DTT
pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 % / Details: 3 second blotting.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Details: Cs corrector
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.37 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 81000. X (nominal), 37037. X (calibrated) / Cs: 0.01 mm / Imaging mode: BRIGHT FIELD / Defocus: 1300.0 - 2500.0 nm / Energy filter: Gatan GIF Quantum LS Imaging Filter / Energy window: 0-20 eV
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: K ( 80.0 - 80.0 K)
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 2382 / Sampling size: 5 microns

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 161000
3D reconstructionAlgorithm: FOURIER SPACE / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Target criteria: correlation coefficient / Refinement space: REAL
Output model

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