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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: EMD-4274
TitleCryoEM structure of E.coli RNA polymerase paused elongation complex without RNA hairpin bound to NusA
Map data
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 / homology
Function and homology information


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 / response to antibiotic / magnesium ion binding / DNA binding ...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 / response to antibiotic / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm
DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase, beta subunit, external 1 domain / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase, alpha subunit, C-terminal / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb2, domain 7 / RNA polymerase subunit, RPB6/omega / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb2, OB-fold ...DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase, beta subunit, external 1 domain / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase, alpha subunit, C-terminal / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb2, domain 7 / RNA polymerase subunit, RPB6/omega / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb2, OB-fold / DNA-directed RNA polymerase, subunit 2 / RPB6/omega subunit-like superfamily / RNA polymerase, beta subunit, protrusion / RNA polymerase, RBP11-like subunit / 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 Rpb1, domain 3 superfamily / RNA polymerase Rpb2, domain 6 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, domain 2 / Bacterial RNA polymerase, alpha chain C terminal domain / RNA polymerase Rpb2, domain 7 / 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 beta subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase, alpha subunit / DNA-directed RNA polymerase, omega 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 Rpb3/Rpb11 dimerisation domain
DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta
Biological speciesEscherichia coli K-12 (bacteria) / synthetic construct (others) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsGuo X / Weixlbaumer A
Funding support France, 1 items
OrganizationGrant numberCountry
European Research CouncilERC starting grant 679734 France
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
History
DepositionJan 26, 2018-
Header (metadata) releaseFeb 28, 2018-
Map releaseMar 7, 2018-
UpdateOct 9, 2019-
Current statusOct 9, 2019Processing site: PDBe / Status: Released

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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

FileDownload / File: emd_4274.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 280 pix.
= 308. Å
1.1 Å/pix.
x 280 pix.
= 308. Å
1.1 Å/pix.
x 280 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 LevelBy AUTHOR: 0.27 / Movie #1: 0.35
Minimum - Maximum-0.50219613 - 1.2147403
Average (Standard dev.)0.005246172 (±0.057818685)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 308.0 Å
α=β=γ: 90.0 °

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/NZ000
NX/NY/NZ224224224
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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Segmentation: #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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