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- EMDB-8585: CryoEM structure of crosslinked E.coli RNA polymerase elongation ... -

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Basic information

Entry
Database: EMDB / ID: 8585
TitleCryoEM structure of crosslinked E.coli RNA polymerase elongation complex
Map dataCryoEM structure of crosslinked E.coli RNA polymerase elongation complex
SampleCrosslinked E.coli RNA polymerase ternary elongation complex
  • (nucleic-acidNucleic acid) x 3
  • (DNA-directed RNA polymerase subunit ...) x 4
  • (ligand) x 2
Function / homologyDNA-directed RNA polymerase, alpha subunit / RPB6/omega subunit-like superfamily / 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 subunit, RPB6/omega / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb2, OB-fold / DNA-directed RNA polymerase, subunit 2 ...DNA-directed RNA polymerase, alpha subunit / RPB6/omega subunit-like superfamily / 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 subunit, RPB6/omega / 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 2 / 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 / RNA polymerase Rpb2, domain 3 / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb2, domain 2 / 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, 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 Rpb2, domain 7 / RNA polymerase complex / DNA-directed RNA polymerase complex / DNA-templated transcription, elongation / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed 5'-3' RNA polymerase activity / 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 / / bacteria /
Methodsingle particle reconstruction / cryo EM / 4.05 Å resolution
AuthorsKang JY / Darst SA
CitationJournal: Elife / Year: 2017
Title: Structural basis of transcription arrest by coliphage HK022 Nun in an RNA polymerase elongation complex.
Authors: Jin Young Kang / Paul Dominic B Olinares / James Chen / Elizabeth A Campbell / Arkady Mustaev / Brian T Chait / Max E Gottesman / Seth A Darst
Abstract: Coliphage HK022 Nun blocks superinfection by coliphage λ by stalling RNA polymerase (RNAP) translocation specifically on λ DNA. To provide a structural framework to understand how Nun blocks RNAP ...Coliphage HK022 Nun blocks superinfection by coliphage λ by stalling RNA polymerase (RNAP) translocation specifically on λ DNA. To provide a structural framework to understand how Nun blocks RNAP translocation, we determined structures of RNAP ternary elongation complexes (TECs) with and without Nun by single-particle cryo-electron microscopy. Nun fits tightly into the TEC by taking advantage of gaps between the RNAP and the nucleic acids. The C-terminal segment of Nun interacts with the RNAP β and β' subunits inside the RNAP active site cleft as well as with nearly every element of the nucleic acid scaffold, essentially crosslinking the RNAP and the nucleic acids to prevent translocation, a mechanism supported by the effects of Nun amino acid substitutions. The nature of Nun interactions inside the RNAP active site cleft suggests that RNAP clamp opening is required for Nun to establish its interactions, explaining why Nun acts on paused TECs.
Validation ReportPDB-ID: 6alf

SummaryFull reportAbout validation report
DateDeposition: Feb 2, 2017 / Header (metadata) release: Apr 5, 2017 / Map release: Apr 5, 2017 / Last update: Apr 5, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6alf
  • Surface level: 0.015
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_8585.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
1.3 Å/pix.
= 332.8 Å
256 pix
1.3 Å/pix.
= 332.8 Å
256 pix
1.3 Å/pix.
= 332.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.3 Å
Density
Contour Level:0.015 (by emdb), 0.015 (movie #1):
Minimum - Maximum-0.03737607 - 0.09016131
Average (Standard dev.)0.00014599922 (0.0035888213)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions256256256
Origin000
Limit255255255
Spacing256256256
CellA=B=C: 332.8 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.31.31.3
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z332.800332.800332.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-152-37
NX/NY/NZ998271
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0370.0900.000

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Supplemental data

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Sample components

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Entire Crosslinked E.coli RNA polymerase ternary elongation complex

EntireName: Crosslinked E.coli RNA polymerase ternary elongation complex
Number of components: 10
MassTheoretical: 393.145 kDa

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Component #1: protein, Crosslinked E.coli RNA polymerase ternary elongation complex

ProteinName: Crosslinked E.coli RNA polymerase ternary elongation complex
Recombinant expression: No
MassTheoretical: 393.145 kDa
SourceSpecies: Escherichia coli
Source (engineered)Expression System: Escherichia coli

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Component #2: nucleic-acid, DNA (29-MER)

Nucleic-acidName: DNA (29-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DG)(DG)(DG)(DC)(DT)(DA)(DC)(DC)(DT)(DC) (DT)(DC)(DC)(DA)(DT)(DG)(DA)(DC)(DG)(DG) (DC)(DG)(DA)(DA)(DT)(DA)(DC)(DC)(DC)
MassTheoretical: 8.840689 kDa
SourceSpecies:

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Component #3: nucleic-acid, DNA (29-MER)

Nucleic-acidName: DNA (29-MER) / Class: DNA / Details: Template DNA / Structure: OTHER / Synthetic: No
Sequence:
(DG)(DG)(DG)(DT)(DA)(DT)(DT)(DC)(DG)(DC) (DC)(DG)(DT)(DG)(DT)(DA)(DC)(DC)(DT)(DC) (DT)(DC)(DC)(DT)(DA)(DG)(DC)(DC)(DC)
MassTheoretical: 8.813646 kDa
SourceSpecies:

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Component #4: nucleic-acid, RNA (5'-R(*GP*CP*A*UP*UP*CP*AP*AP*AP*GP*CP*GP*GP*AP...

Nucleic-acidName: RNA (5'-R(*GP*CP*A*UP*UP*CP*AP*AP*AP*GP*CP*GP*GP*AP*GP*AP*GP*GP*UP*A)-3')
Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
GCAUUCAAAG CGGAGAGGUA
MassTheoretical: 6.509968 kDa
SourceSpecies:

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Component #5: protein, DNA-directed RNA polymerase subunit alpha

ProteinName: DNA-directed RNA polymerase subunit alpha / Recombinant expression: No
MassTheoretical: 26.459125 kDa
SourceSpecies:
Source (engineered)Expression System: Escherichia coli O157:H7

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Component #6: protein, DNA-directed RNA polymerase subunit beta

ProteinName: DNA-directed RNA polymerase subunit beta / Recombinant expression: No
MassTheoretical: 150.820875 kDa
SourceSpecies:
Source (engineered)Expression System: Escherichia coli O45:K1 (strain S88 / ExPEC)
Strain: S88 / ExPEC

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Component #7: protein, DNA-directed RNA polymerase subunit beta'

ProteinName: DNA-directed RNA polymerase subunit beta' / Recombinant expression: No
MassTheoretical: 155.366781 kDa
SourceSpecies:
Source (engineered)Expression System: Escherichia coli O157:H7

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

ProteinName: DNA-directed RNA polymerase subunit omega / Recombinant expression: No
MassTheoretical: 9.094239 kDa
SourceSpecies:
Source (engineered)Expression System: Escherichia coli O45:K1 (strain S88 / ExPEC)
Strain: S88 / ExPEC

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Component #9: ligand, MAGNESIUM ION

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

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Component #10: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 6.540905 MDa
SourceSpecies:

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 3 mg/ml / pH: 8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 295 K / Humidity: 100 %

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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: 85.2 e/Å2 / Illumination mode: FLOOD BEAM
LensCs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 800 - 2400 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 2000

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 83800
3D reconstructionSoftware: RELION / CTF correction: CTF parameters were estimated by CTFFIND4 / Resolution: 4.05 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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