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- EMDB-0001: Cryo-EM structure of bacterial RNA polymerase-sigma54 holoenzyme ... -

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Basic information

Entry
Database: EMDB / ID: 0001
TitleCryo-EM structure of bacterial RNA polymerase-sigma54 holoenzyme transcription open complex
Map data
SampleCryo-EM structure of bacterial RNA polymerase-sigma54 holoenzyme transcription open complex
  • DNA-directed RNA polymerasePolymerase
  • (RNA polymerase sigma-54 ...) x 2
  • DNA
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • (nucleic-acidNucleic acid) x 2
Function / homologyRNA polymerase subunit, RPB6/omega / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase, alpha subunit, C-terminal / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb2, OB-fold / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, beta subunit, external 1 domain ...RNA polymerase subunit, RPB6/omega / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase, alpha subunit, C-terminal / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb2, OB-fold / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, beta subunit, external 1 domain / RPB6/omega subunit-like superfamily / RNA polymerase, RBP11-like subunit / DNA-directed RNA polymerase, insert domain superfamily / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb2, domain 3 / RNA polymerase sigma-54 factor, core-binding domain superfamily / Sigma-54 factor, Activator interacting domain (AID) / 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-54, DNA binding domain / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 2 / RNA polymerase beta subunit / RNA polymerase Rpb2, domain 3 / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb1, domain 3 / RNA polymerase, subunit omega/K/RPB6 / RNA polymerases beta chain signature. / Sigma-54 factors family signature 2. / Sigma-54 factors family signature 1. / RNA polymerase beta subunit external 1 domain / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb2, domain 2 / RNA polymerase, alpha subunit / DNA-directed RNA polymerase, omega subunit / RNA polymerase sigma factor 54 / RNA polymerase, N-terminal / RNA polymerase sigma factor 54, core-binding domain / RNA polymerase Rpb2, domain 7 / RNA polymerase sigma factor 54, DNA-binding / 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 / Sigma-54 factor, core binding domain / RNA polymerase complex / DNA-directed RNA polymerase complex / bacterial-type RNA polymerase transcriptional activator activity, sequence-specific DNA binding / DNA-templated transcription, elongation / sigma factor activity / ribonucleoside binding / DNA-templated transcription, initiation / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / transcription, DNA-templated / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm / RNA polymerase sigma-54 factor / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta
Function and homology information
SourceEscherichia coli K-12 (bacteria) / Klebsiella pneumoniae (bacteria) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / 3.4 Å resolution
AuthorsGlyde R / Ye FZ / Zhang XD
CitationJournal: Mol. Cell / Year: 2018
Title: Structures of Bacterial RNA Polymerase Complexes Reveal the Mechanism of DNA Loading and Transcription Initiation.
Authors: Robert Glyde / Fuzhou Ye / Milija Jovanovic / Ioly Kotta-Loizou / Martin Buck / Xiaodong Zhang
Abstract: Gene transcription is carried out by multi-subunit RNA polymerases (RNAPs). Transcription initiation is a dynamic multi-step process that involves the opening of the double-stranded DNA to form a ...Gene transcription is carried out by multi-subunit RNA polymerases (RNAPs). Transcription initiation is a dynamic multi-step process that involves the opening of the double-stranded DNA to form a transcription bubble and delivery of the template strand deep into the RNAP for RNA synthesis. Applying cryoelectron microscopy to a unique transcription system using σ (σ), the major bacterial variant sigma factor, we capture a new intermediate state at 4.1 Å where promoter DNA is caught at the entrance of the RNAP cleft. Combining with new structures of the open promoter complex and an initial de novo transcribing complex at 3.4 and 3.7 Å, respectively, our studies reveal the dynamics of DNA loading and mechanism of transcription bubble stabilization that involves coordinated, large-scale conformational changes of the universally conserved features within RNAP and DNA. In addition, our studies reveal a novel mechanism of strand separation by σ.
Validation ReportPDB-ID: 6gh5

SummaryFull reportAbout validation report
DateDeposition: May 4, 2018 / Header (metadata) release: Jun 6, 2018 / Map release: Jul 4, 2018 / Last update: Jul 4, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6gh5
  • Surface level: 0.02
  • 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_0001.map.gz (map file in CCP4 format, 67109 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
256 pix
1.06 Å/pix.
= 271.36 Å
256 pix
1.06 Å/pix.
= 271.36 Å
256 pix
1.06 Å/pix.
= 271.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density
Contour Level:0.018 (by emdb), 0.02 (movie #1):
Minimum - Maximum-0.10788548 - 0.22688344
Average (Standard dev.)0.000012941896 (0.0077065425)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions256256256
Origin0.0.0.
Limit255.255.255.
Spacing256256256
CellA=B=C: 1.0 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z271.360271.360271.360
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1080.2270.000

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Supplemental data

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Sample components

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Entire Cryo-EM structure of bacterial RNA polymerase-sigma54 holoenzyme ...

EntireName: Cryo-EM structure of bacterial RNA polymerase-sigma54 holoenzyme transcription open complex
Number of components: 11

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Component #1: protein, Cryo-EM structure of bacterial RNA polymerase-sigma54 ho...

ProteinName: Cryo-EM structure of bacterial RNA polymerase-sigma54 holoenzyme transcription open complex
Recombinant expression: No
MassTheoretical: 490 kDa

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Component #2: protein, DNA-directed RNA polymerase

ProteinName: DNA-directed RNA polymerasePolymerase / Recombinant expression: No
SourceSpecies: Escherichia coli K-12 (bacteria)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Component #3: protein, RNA polymerase sigma-54 factor

ProteinName: RNA polymerase sigma-54 factor / Recombinant expression: No
SourceSpecies: Klebsiella pneumoniae (bacteria)
Source (engineered)Expression System: Escherichia coli K-12 (bacteria)

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Component #4: protein, DNA

ProteinName: DNA / Recombinant expression: No
SourceSpecies: Klebsiella pneumoniae (bacteria)
Source (engineered)Expression System: synthetic construct (others)

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Component #5: 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)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Component #6: 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)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Component #7: 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 (strain K12) (bacteria)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Component #8: 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)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Component #9: protein, RNA polymerase sigma-54 factor,RNA polymerase sigma-54 f...

ProteinName: RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 54.723082 kDa
SourceSpecies: Klebsiella pneumoniae (bacteria)
Source (engineered)Expression System: Escherichia coli K-12 (bacteria)

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Component #10: nucleic-acid, nifH promoter template DNA

Nucleic-acidName: nifH promoter template DNA / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DA)(DC)(DA)(DT)(DG)(DA)(DA)(DT)(DG)(DC) (DG)(DC)(DA)(DA)(DC)(DA)(DG)(DC)(DA)(DT) (DG)(DC)(DG)(DC)(DG)(DC)(DC)(DC)(DA)(DG) (DG)(DG)(DC)(DT)(DG)(DA)(DT)(DC)(DG)(DT) (DG)(DC)(DA)(DA)(DA)(DA)(DG)(DT)(DC)(DG) (DT)(DG)(DC)(DC)(DA)(DG)(DC)(DC)(DG)(DT) (DC)(DT)(DC)
MassTheoretical: 19.40441 kDa
SourceSpecies: Klebsiella pneumoniae (bacteria)

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Component #11: nucleic-acid, nifH promoter non-template DNA

Nucleic-acidName: nifH promoter non-template DNA / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DG)(DA)(DG)(DA)(DC)(DG)(DG)(DC)(DT)(DG) (DG)(DC)(DA)(DC)(DG)(DA)(DC)(DT)(DT)(DT) (DT)(DG)(DC)(DA)(DC)(DT)(DC)(DG)(DA)(DC) (DT)(DA)(DA)(DA)(DG)(DG)(DG)(DG)(DC)(DG) (DC)(DG)(DC)(DA)(DT)(DG)(DC)(DT)(DG)(DT) (DT)(DG)(DC)(DG)(DC)(DA)(DT)(DT)(DC)(DA) (DT)(DG)(DT)
MassTheoretical: 19.487436 kDa
SourceSpecies: Klebsiella pneumoniae (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.5 mg/ml / pH: 8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 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: 45 e/Å2 / Illumination mode: OTHER
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: 79678
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Refinement space: REAL
Output model

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