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- EMDB-4274: CryoEM structure of E.coli RNA polymerase paused elongation compl... -

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Basic information

Entry
Database: EMDB / ID: 4274
TitleCryoEM structure of E.coli RNA polymerase paused elongation complex without RNA hairpin bound to NusA
Map dataE. coli RNA polymerase paused elongation complex bound to NusA without density for RNA hairpin
SampleCryo-EM structure of E. coli RNA polymerase paused elongation complex without RNA hairpin bound to NusA
  • RNA polymerase and NusA
  • Nucleic acidsNucleic acid
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • (nucleic-acidNucleic acid) x 3
  • (ligand) x 2
Function / homologyRNA polymerase subunit, RPB6/omega / RPB6/omega subunit-like 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 / RNA polymerase beta subunit / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb2, OB-fold / DNA-directed RNA polymerase, subunit 2 ...RNA polymerase subunit, RPB6/omega / RPB6/omega subunit-like 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 / RNA polymerase beta 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, RBP11-like subunit / RNA polymerase Rpb2, domain 3 / 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 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 / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb2, domain 2 / DNA-directed RNA polymerase, omega subunit / RNA polymerases beta chain signature. / 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 Rpb2, domain 2 / RNA polymerase, alpha subunit / RNA polymerase, subunit omega/K/RPB6 / RNA polymerase, N-terminal / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 7 / RNA polymerase complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein-containing complex assembly / protein dimerization activity / transcription, DNA-templated / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm / 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) / synthetic construct (others) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / 4.1 Å resolution
AuthorsGuo X / Weixlbaumer A
CitationJournal: Mol. Cell / Year: 2018
Title: Structural Basis for NusA Stabilized Transcriptional Pausing.
Authors: Xieyang Guo / Alexander G Myasnikov / James Chen / Corinne Crucifix / Gabor Papai / Maria Takacs / Patrick Schultz / Albert Weixlbaumer
Abstract: Transcriptional pausing by RNA polymerases (RNAPs) is a key mechanism to regulate gene expression in all kingdoms of life and is a prerequisite for transcription termination. The essential bacterial ...Transcriptional pausing by RNA polymerases (RNAPs) is a key mechanism to regulate gene expression in all kingdoms of life and is a prerequisite for transcription termination. The essential bacterial transcription factor NusA stimulates both pausing and termination of transcription, thus playing a central role. Here, we report single-particle electron cryo-microscopy reconstructions of NusA bound to paused E. coli RNAP elongation complexes with and without a pause-enhancing hairpin in the RNA exit channel. The structures reveal four interactions between NusA and RNAP that suggest how NusA stimulates RNA folding, pausing, and termination. An asymmetric translocation intermediate of RNA and DNA converts the active site of the enzyme into an inactive state, providing a structural explanation for the inhibition of catalysis. Comparing RNAP at different stages of pausing provides insights on the dynamic nature of the process and the role of NusA as a regulatory factor.
Validation ReportPDB-ID: 6flp

SummaryFull reportAbout validation report
DateDeposition: Jan 26, 2018 / Header (metadata) release: Feb 28, 2018 / Map release: Mar 7, 2018 / Last update: Mar 21, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.35
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.35
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6flp
  • Surface level: 0.35
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_4274.map.gz (map file in CCP4 format, 87809 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
280 pix
1.1 Å/pix.
= 308. Å
280 pix
1.1 Å/pix.
= 308. Å
280 pix
1.1 Å/pix.
= 308. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density
Contour Level:0.027 (by author), 0.35 (movie #1):
Minimum - Maximum-0.06597029 - 0.102163516
Average (Standard dev.)0.000042178486 (0.0046332427)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions280280280
Origin0.00.00.0
Limit279.0279.0279.0
Spacing280280280
CellA=B=C: 308.0 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z308.000308.000308.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-200-200-200
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.5021.2150.005

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Supplemental data

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Mask #1

Fileemd_4274_msk_1.map
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms

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Sample components

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Entire Cryo-EM structure of E. coli RNA polymerase paused elongation com...

EntireName: Cryo-EM structure of E. coli RNA polymerase paused elongation complex without RNA hairpin bound to NusA
Number of components: 12

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Component #1: protein, Cryo-EM structure of E. coli RNA polymerase paused elong...

ProteinName: Cryo-EM structure of E. coli RNA polymerase paused elongation complex without RNA hairpin bound to NusA
Recombinant expression: No

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Component #2: protein, RNA polymerase and NusA

ProteinName: RNA polymerase and NusA / Recombinant expression: No
SourceSpecies: Escherichia coli K-12 (bacteria)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #3: protein, Nucleic acids

ProteinName: Nucleic acidsNucleic acid / Recombinant expression: No
SourceSpecies: synthetic construct (others)
Source (engineered)Expression System: synthetic construct (others)

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Component #4: 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 BL21(DE3) (bacteria)

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Component #5: 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 BL21(DE3) (bacteria)

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Component #6: 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) / Strain: K12
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #7: 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 BL21(DE3) (bacteria)

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Component #8: nucleic-acid, DNA (30-MER)

Nucleic-acidName: DNA (30-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DG)(DG)(DG)(DA)(DC)(DG)(DT)(DA)(DC)(DT) (DG)(DA)(DC)(DC)(DG)(DC)(DG)(DG)(DA)(DA) (DG)(DA)(DG)(DA)(DT)(DT)(DC)(DA)(DG)(DA) (DG)
MassTheoretical: 9.676235 kDa
SourceSpecies: synthetic construct (others)

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

Nucleic-acidName: RNA (5'-R(P*GP*AP*UP*GP*UP*GP*UP*GP*CP*U)-3') / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
GAUGUGUGCU
MassTheoretical: 3.194917 kDa
SourceSpecies: synthetic construct (others)

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Component #10: nucleic-acid, DNA (39-MER)

Nucleic-acidName: DNA (39-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DC)(DT)(DC)(DT)(DG)(DA)(DA)(DT)(DC)(DT) (DC)(DT)(DT)(DC)(DC)(DA)(DG)(DC)(DA)(DC) (DA)(DC)(DA)(DT)(DC)(DG)(DG)(DT)(DC)(DA) (DG)(DT)(DA)(DC)(DG)(DT)(DC)(DC)(DC)
MassTheoretical: 11.815586 kDa
SourceSpecies: synthetic construct (others)

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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: 8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 283.15 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: 53 e/Å2 / Illumination mode: SPOT SCAN
LensMagnification: 105000.0 X (nominal) / Cs: 0.001 mm / Imaging mode: BRIGHT FIELD / Defocus: 800.0 - 3200.0 nm / Energy filter: GIF Quantum ER / Energy window: 0-20 eV
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 4957

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 65966
3D reconstructionSoftware: cryoSPARC / Resolution: 4.1 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
Output model

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