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- EMDB-14208: Cryo-EM structure of RNA polymerase-sigma54 holoenzyme with promo... -

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Basic information

Entry
Database: EMDB / ID: EMD-14208
TitleCryo-EM structure of RNA polymerase-sigma54 holoenzyme with promoter DNA and transcription activator PspF intermedate complex
Map dataLocal filtered map
Sample
  • Complex: Cryo-EM structure of RNA polymerase-sigma54 holoenzyme with promoter DNA and transcription activator PspF intermedate complex
KeywordsCryo EM / RNA polymerase / transcription intermediate complex / sigma54 / TRANSCRIPTION
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsYe FZ / Zhang XD
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/N007816/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/P007503/1 United Kingdom
CitationJournal: Sci Adv / Year: 2022
Title: Mechanisms of DNA opening revealed in AAA+ transcription complex structures.
Authors: Fuzhou Ye / Forson Gao / Xiaojiao Liu / Martin Buck / Xiaodong Zhang /
Abstract: Gene transcription is carried out by RNA polymerase (RNAP) and requires the conversion of the initial closed promoter complex, where DNA is double stranded, to a transcription-competent open promoter ...Gene transcription is carried out by RNA polymerase (RNAP) and requires the conversion of the initial closed promoter complex, where DNA is double stranded, to a transcription-competent open promoter complex, where DNA is opened up. In bacteria, RNAP relies on σ factors for its promoter specificities. Using a special form of sigma factor (σ), which forms a stable closed complex and requires its activator that belongs to the AAA+ ATPases (ATPases associated with diverse cellular activities), we obtained cryo-electron microscopy structures of transcription initiation complexes that reveal a previously unidentified process of DNA melting opening. The σ amino terminus threads through the locally opened up DNA and then becomes enclosed by the AAA+ hexameric ring in the activator-bound intermediate complex. Our structures suggest how ATP hydrolysis by the AAA+ activator could remove the σ inhibition while helping to open up DNA, using σ amino-terminal peptide as a pry bar.
History
DepositionJan 27, 2022-
Header (metadata) releaseNov 9, 2022-
Map releaseNov 9, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14208.png

Downloads & links

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Map

FileDownload / File: emd_14208.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal filtered map
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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.014
Minimum - Maximum-0.09395847 - 0.15217389
Average (Standard dev.)0.000493699 (±0.004607518)
SymmetrySpace group: 1
Details

EMDB XML:

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

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Supplemental data

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Sample components

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Entire : Cryo-EM structure of RNA polymerase-sigma54 holoenzyme with promo...

EntireName: Cryo-EM structure of RNA polymerase-sigma54 holoenzyme with promoter DNA and transcription activator PspF intermedate complex
Components
  • Complex: Cryo-EM structure of RNA polymerase-sigma54 holoenzyme with promoter DNA and transcription activator PspF intermedate complex

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Supramolecule #1: Cryo-EM structure of RNA polymerase-sigma54 holoenzyme with promo...

SupramoleculeName: Cryo-EM structure of RNA polymerase-sigma54 holoenzyme with promoter DNA and transcription activator PspF intermedate complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Details: complex composition: RNA polymease plus promoter DNA and transcription activator PspF
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 700 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris
8.0 mMC32H58N2O7SCHAPSO
150.0 mMKCLpotassium chloride
10.0 mMMgCL2Magnesium chloride

Details: 20 mM Tris-HCl pH 8.0, 150 mM KCl, 10 mM MgCl2,8 mM CHAPSO
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThe sample was mxied in vitro in an order to form complex

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 14780 / Average exposure time: 4.1 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.4000000000000001 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2500000
Startup modelType of model: EMDB MAP
EMDB ID:

3696

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. Relion3.1) / Number images used: 23354
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. Relion3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. Relion3.1)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. Relion3.1)

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Atomic model buiding 1

Initial modelPDB ID:

5nss


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT

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