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- EMDB-3697: Cryo-EM structure of RNA polymerase-sigma54 holoenzyme with promo... -

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Basic information

Entry
Database: EMDB / ID: EMD-3697
TitleCryo-EM structure of RNA polymerase-sigma54 holoenzyme with promoter DNA and transcription activator PspF intermedate complex
Map data
Sample
  • Complex: Cryo-EM structure of RNA polymerase -sigma54 holoenzyme with promoter DNA and transcription activator PspF
    • Complex: Escherichia coli core RNA polymerase
    • Complex: PspF transcriptional activator
    • Complex: Sigma-54 transcription initiation factor
    • Complex: Sigma-54 promoter DNA
Function / homology
Function and homology information


regulation of cellular response to stress / nitrogen fixation / RNA polymerase complex / submerged biofilm formation / DNA-binding transcription activator activity / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / sigma factor activity ...regulation of cellular response to stress / nitrogen fixation / RNA polymerase complex / submerged biofilm formation / DNA-binding transcription activator activity / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / sigma factor activity / bacterial-type flagellum-dependent cell motility / phosphorelay signal transduction system / nitrate assimilation / nucleotidyltransferase activity / transcription elongation factor complex / DNA-directed RNA polymerase complex / regulation of DNA-templated transcription elongation / transcription antitermination / cell motility / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / protein-containing complex assembly / intracellular iron ion homeostasis / transcription regulator complex / sequence-specific DNA binding / protein dimerization activity / response to antibiotic / negative regulation of DNA-templated transcription / regulation of DNA-templated transcription / positive regulation of DNA-templated transcription / magnesium ion binding / ATP hydrolysis activity / DNA binding / zinc ion binding / ATP binding / membrane / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Transcription activator PspF / Sigma-54 factors family signature 1. / Sigma-54 factors family profile. / RNA polymerase sigma factor 54, core-binding domain / RNA polymerase sigma factor 54, DNA-binding / RNA polymerase sigma-54 factor, core-binding domain superfamily / Sigma-54 factor, Activator interacting domain (AID) / Sigma-54, DNA binding domain / Sigma-54 factor, core binding domain / Sigma-54 factors family signature 2. ...Transcription activator PspF / Sigma-54 factors family signature 1. / Sigma-54 factors family profile. / RNA polymerase sigma factor 54, core-binding domain / RNA polymerase sigma factor 54, DNA-binding / RNA polymerase sigma-54 factor, core-binding domain superfamily / Sigma-54 factor, Activator interacting domain (AID) / Sigma-54, DNA binding domain / Sigma-54 factor, core binding domain / Sigma-54 factors family signature 2. / RNA polymerase sigma factor 54 / Sigma-54 interaction domain, conserved site / Sigma-54 interaction domain C-terminal part signature. / Sigma-54 interaction domain, ATP-binding site 2 / Sigma-54 interaction domain ATP-binding region B signature. / Sigma-54 interaction domain profile. / Sigma-54 interaction domain / RNA polymerase sigma factor 54 interaction domain / DNA binding HTH domain, Fis-type / Bacterial regulatory protein, Fis family / DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase beta subunit external 1 domain / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, OB-fold / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerases beta chain signature. / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 6 / Homeobox-like domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
RNA polymerase sigma-54 factor / RNA polymerase sigma-54 factor / : / RNA polymerase sigma-54 factor / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / Psp operon transcriptional activator
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria) / Klebsiella pneumoniae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.9 Å
AuthorsGlyde R / Ye FZ / Darbari VC / Zhang N / Buck M / Zhang XD
CitationJournal: Mol Cell / Year: 2017
Title: Structures of RNA Polymerase Closed and Intermediate Complexes Reveal Mechanisms of DNA Opening and Transcription Initiation.
Authors: Robert Glyde / Fuzhou Ye / Vidya Chandran Darbari / Nan Zhang / Martin Buck / Xiaodong Zhang /
Abstract: Gene transcription is carried out by RNA polymerases (RNAPs). For transcription to occur, the closed promoter complex (RPc), where DNA is double stranded, must isomerize into an open promoter complex ...Gene transcription is carried out by RNA polymerases (RNAPs). For transcription to occur, the closed promoter complex (RPc), where DNA is double stranded, must isomerize into an open promoter complex (RPo), where the DNA is melted out into a transcription bubble and the single-stranded template DNA is delivered to the RNAP active site. Using a bacterial RNAP containing the alternative σ factor and cryoelectron microscopy, we determined structures of RPc and the activator-bound intermediate complex en route to RPo at 3.8 and 5.8 Å. Our structures show how RNAP-σ interacts with promoter DNA to initiate the DNA distortions required for transcription bubble formation, and how the activator interacts with RPc, leading to significant conformational changes in RNAP and σ that promote RPo formation. We propose that DNA melting is an active process initiated in RPc and that the RNAP conformations of intermediates are significantly different from that of RPc and RPo.
History
DepositionApr 27, 2017-
Header (metadata) releaseJun 28, 2017-
Map releaseJun 28, 2017-
UpdateNov 28, 2018-
Current statusNov 28, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0039
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0039
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3697.png

Downloads & links

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Map

FileDownload / File: emd_3697.map.gz / Format: CCP4 / Size: 76.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy EMDB: 0.00576 / Movie #1: 0.0039
Minimum - Maximum-0.0073748953 - 0.02324747
Average (Standard dev.)0.00017240495 (±0.0009918918)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions272272272
Spacing272272272
CellA=B=C: 285.59998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z272272272
origin x/y/z0.0000.0000.000
length x/y/z285.600285.600285.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS272272272
D min/max/mean-0.0070.0230.000

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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 prom...

EntireName: Cryo-EM structure of RNA polymerase -sigma54 holoenzyme with promoter DNA and transcription activator PspF
Components
  • Complex: Cryo-EM structure of RNA polymerase -sigma54 holoenzyme with promoter DNA and transcription activator PspF
    • Complex: Escherichia coli core RNA polymerase
    • Complex: PspF transcriptional activator
    • Complex: Sigma-54 transcription initiation factor
    • Complex: Sigma-54 promoter DNA

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

SupramoleculeName: Cryo-EM structure of RNA polymerase -sigma54 holoenzyme with promoter DNA and transcription activator PspF
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Supramolecule #2: Escherichia coli core RNA polymerase

SupramoleculeName: Escherichia coli core RNA polymerase / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Supramolecule #3: PspF transcriptional activator

SupramoleculeName: PspF transcriptional activator / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Supramolecule #4: Sigma-54 transcription initiation factor

SupramoleculeName: Sigma-54 transcription initiation factor / type: complex / ID: 4 / Parent: 1
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Supramolecule #5: Sigma-54 promoter DNA

SupramoleculeName: Sigma-54 promoter DNA / type: complex / ID: 5 / Parent: 1
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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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

BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.25 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 89397

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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