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- EMDB-21920: Cryo-EM structure of Bacillus subtilis RNA Polymerase elongation ... -

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Basic information

Entry
Database: EMDB / ID: EMD-21920
TitleCryo-EM structure of Bacillus subtilis RNA Polymerase elongation complex
Map data
SampleDNA-directed RNA polymerase elongation complex
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 5
  • DNA, RNA
  • (nucleic-acidNucleic acid) x 3
  • (ligand) x 2
Function / homology
Function and homology information


ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / transcription, DNA-templated / protein dimerization activity / response to antibiotic / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
DNA-directed RNA polymerase, omega subunit / RNA polymerase, alpha subunit, C-terminal / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / Bacterial RNA polymerase, alpha chain C terminal domain / RNA polymerase beta subunit external 1 domain / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, beta subunit, external 1 domain / DNA-directed RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb6 ...DNA-directed RNA polymerase, omega subunit / RNA polymerase, alpha subunit, C-terminal / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / Bacterial RNA polymerase, alpha chain C terminal domain / RNA polymerase beta subunit external 1 domain / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, beta subunit, external 1 domain / DNA-directed RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb6 / RPB6/omega subunit-like superfamily / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / : / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 4 / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 3 / RNA polymerase, N-terminal / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb2, domain 2 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase Rpb2, domain 2 / DNA-directed RNA polymerase, insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase Rpb3/RpoA insert domain / RNA polymerases D / DNA-directed RNA polymerase, insert domain superfamily / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, domain 3 / RNA polymerases beta chain signature. / RNA polymerase Rpb2, domain 6 / DNA-directed RNA polymerase, subunit 2 / RNA polymerase Rpb2, OB-fold / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / RNA polymerase Rpb2, domain 7 / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily
Similarity search - Domain/homology
DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit beta'
Similarity search - Component
Biological speciesBacillus subtilis (bacteria) / Escherichia coli BL21(DE3) (bacteria) / Bacillus subtilis (strain 168) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.36 Å
AuthorsNewing T / Tolun G / Oakley AJ
CitationJournal: Nat Commun / Year: 2020
Title: Molecular basis for RNA polymerase-dependent transcription complex recycling by the helicase-like motor protein HelD.
Authors: Timothy P Newing / Aaron J Oakley / Michael Miller / Catherine J Dawson / Simon H J Brown / James C Bouwer / Gökhan Tolun / Peter J Lewis /
Abstract: In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Gram- ...In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Gram-positive bacteria contain a transcription factor HelD that is able to remove and recycle stalled complexes, but it was not known how it performed this function. Here, using single particle cryo-electron microscopy, we have determined the structures of Bacillus subtilis RNA polymerase (RNAP) elongation and HelD complexes, enabling analysis of the conformational changes that occur in RNAP driven by HelD interaction. HelD has a 2-armed structure which penetrates deep into the primary and secondary channels of RNA polymerase. One arm removes nucleic acids from the active site, and the other induces a large conformational change in the primary channel leading to removal and recycling of the stalled polymerase, representing a novel mechanism for recycling transcription complexes in bacteria.
History
DepositionMay 6, 2020-
Header (metadata) releaseNov 18, 2020-
Map releaseNov 18, 2020-
UpdateJan 6, 2021-
Current statusJan 6, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.55
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.55
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6wvj
  • Surface level: 1.55
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_21920.map.gz / Format: CCP4 / Size: 20.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.84 Å/pix.
x 188 pix.
= 157.92 Å
0.84 Å/pix.
x 172 pix.
= 144.48 Å
0.84 Å/pix.
x 166 pix.
= 139.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 0.84 Å
Density
Contour LevelBy AUTHOR: 1.55 / Movie #1: 1.55
Minimum - Maximum-5.7642713 - 10.3556385
Average (Standard dev.)-0.02620416 (±0.7741332)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin656155
Dimensions172166188
Spacing188172166
CellA: 157.92 Å / B: 144.48 Å / C: 139.44 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.840.840.84
M x/y/z188172166
origin x/y/z0.0000.0000.000
length x/y/z157.920144.480139.440
α/β/γ90.00090.00090.000
start NX/NY/NZ556561
NX/NY/NZ188172166
MAP C/R/S321
start NC/NR/NS616555
NC/NR/NS166172188
D min/max/mean-5.76410.356-0.026

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Supplemental data

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Sample components

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Entire DNA-directed RNA polymerase elongation complex

EntireName: DNA-directed RNA polymerase elongation complex / Number of Components: 12

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Component #1: protein, DNA-directed RNA polymerase elongation complex

ProteinName: DNA-directed RNA polymerase elongation complex / Recombinant expression: No
MassExperimental: 344.2 kDa

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Component #2: protein, DNA-directed RNA polymerase subunit alpha, DNA-directed ...

ProteinName: DNA-directed RNA polymerase subunit alpha, DNA-directed RNA polymerase subunit beta, DNA-directed RNA polymerase subunit beta', DNA-directed RNA polymerase subunit omega/DNA ComplexPolymerase
Recombinant expression: No
SourceSpecies: Bacillus subtilis (bacteria)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria) / Vector: pNG1256

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Component #3: protein, DNA, RNA

ProteinName: DNA, RNA / Recombinant expression: No
SourceSpecies: Escherichia coli BL21(DE3) (bacteria)

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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: 34.842387 kDa
SourceSpecies: Bacillus subtilis (strain 168) (bacteria) / Strain: 168
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: 133.847938 kDa
SourceSpecies: Bacillus subtilis (bacteria)
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: 134.444953 kDa
SourceSpecies: Bacillus subtilis (bacteria)
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: 7.766921 kDa
SourceSpecies: Bacillus subtilis (bacteria)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #8: nucleic-acid, DNA (5'-D(*TP*GP*TP*CP*GP*GP*GP*CP*GP*TP*CP*CP*GP*C...

nucleic acidName: DNA (5'-D(*TP*GP*TP*CP*GP*GP*GP*CP*GP*TP*CP*CP*GP*CP*GP*CP*GP*CP*C)-3')
Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DT)(DG)(DT)(DC)(DG)(DG)(DG)(DC)(DG)(DT) (DC)(DC)(DG)(DC)(DG)(DC)(DG)(DC)(DC)
MassTheoretical: 5.814721 kDa
SourceSpecies: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)

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

nucleic acidName: RNA (5'-R(P*GP*GP*CP*GP*CP*GP*CP*G)-3') / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
GGCGCGCG
MassTheoretical: 2.596617 kDa
SourceSpecies: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)

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Component #10: nucleic-acid, DNA (5'-D(P*AP*CP*GP*CP*CP*CP*GP*AP*CP*A)-3')

nucleic acidName: DNA (5'-D(P*AP*CP*GP*CP*CP*CP*GP*AP*CP*A)-3') / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DA)(DC)(DG)(DC)(DC)(DC)(DG)(DA)(DC)(DA)
MassTheoretical: 2.998984 kDa
SourceSpecies: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)

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Component #11: ligand, ZINC ION

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

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Component #12: ligand, MAGNESIUM ION

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

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Experimental details

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Sample preparation

SpecimenSpecimen State: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 2.5 mg/mL / pH: 7.8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen Name: ETHANE / Temperature: 298 K / Humidity: 100 %
Details: Sample loading volume ranged between 2 and 3 microlitres. Samples were blotted for 5 seconds prior to vitrification..

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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: 52.2 e/Å2 / Illumination Mode: FLOOD BEAM
LensMagnification: 59500 X (calibrated) / Cs: 2.7 mm / Imaging Mode: BRIGHT FIELD / Defocus: 600.0 - 2800.0 nm / Energy Filter: GIF Quantum LS
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: 5185

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Image processing

ProcessingMethod: single particle reconstruction / Applied Symmetry: C1 (asymmetric) / Number of Projections: 58854
3D reconstructionSoftware: RELION / Resolution: 3.36 Å / Resolution Method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement space: REAL
Output model

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