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- PDB-9q91: CryoEM structure of bacterial transcription intermediate complex ... -

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Basic information

Entry
Database: PDB / ID: 9q91
TitleCryoEM structure of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 6
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • Non-template DNA (34-MER)
  • Psp operon transcriptional activator
  • RNA polymerase sigma-54 factor
  • Template DNA (34-MER)
KeywordsTRANSCRIPTION / sigma factor / complex / AAA+ / DNA-binding protein
Function / homology
Function and homology information


regulation of cellular response to stress / DNA-binding transcription activator activity / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / bacterial-type RNA polymerase core enzyme binding / regulation of DNA-templated transcription initiation / phosphorelay signal transduction system / sigma factor activity ...regulation of cellular response to stress / DNA-binding transcription activator activity / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / bacterial-type RNA polymerase core enzyme binding / regulation of DNA-templated transcription initiation / phosphorelay signal transduction system / sigma factor activity / bacterial-type flagellum assembly / bacterial-type flagellum-dependent cell motility / nitrate assimilation / nucleotidyltransferase activity / DNA-directed RNA polymerase complex / transcription elongation factor complex / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription initiation / cell motility / ribonucleoside binding / : / : / : / : / : / : / DNA-directed RNA polymerase / response to heat / protein-containing complex assembly / transcription regulator complex / sequence-specific DNA binding / intracellular iron ion homeostasis / 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 / identical protein binding / membrane / cytosol / cytoplasm
Similarity search - Function
Transcription activator PspF / Sigma-54 factors family signature 1. / 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. / Sigma-54 factors family profile. ...Transcription activator PspF / Sigma-54 factors family signature 1. / 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. / Sigma-54 factors family profile. / 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 / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / 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 / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / 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. / Homeobox-like domain superfamily / 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 / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA / DNA (> 10) / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.2 Å
AuthorsGao, F. / Zhang, X.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC) United Kingdom
CitationJournal: To Be Published
Title: Subunit specialisation in AAA+ proteins and substrate unfolding during transcription complex remodelling
Authors: Gao, F. / Ye, F. / Buck, M. / Zhang, X.
History
DepositionFeb 26, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 2, 2025Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
1: Psp operon transcriptional activator
2: Psp operon transcriptional activator
3: Psp operon transcriptional activator
4: Psp operon transcriptional activator
A: DNA-directed RNA polymerase subunit alpha
C: DNA-directed RNA polymerase subunit beta
E: DNA-directed RNA polymerase subunit omega
N: Non-template DNA (34-MER)
T: Template DNA (34-MER)
5: Psp operon transcriptional activator
6: Psp operon transcriptional activator
M: RNA polymerase sigma-54 factor
B: DNA-directed RNA polymerase subunit alpha
D: DNA-directed RNA polymerase subunit beta'


Theoretical massNumber of molelcules
Total (without water)640,41314
Polymers640,41314
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 7 molecules 123456M

#1: Protein
Psp operon transcriptional activator / Phage shock protein F


Mass: 29390.668 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: pspF, ycjB, b1303, JW1296 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P37344
#7: Protein RNA polymerase sigma-54 factor


Mass: 53722.402 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: NCTC13443_06667 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: A0A377VEN9

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DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules ABCED

#2: Protein DNA-directed RNA polymerase subunit alpha / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 36558.680 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpoA, pez, phs, sez, b3295, JW3257 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta


Mass: 150691.750 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpoB / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A8V2
#4: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 10249.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpoZ, b3649, JW3624 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A800, DNA-directed RNA polymerase
#8: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 155366.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpoC, tabB, b3988, JW3951 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A8T7, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules NT

#5: DNA chain Non-template DNA (34-MER)


Mass: 10420.691 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli K-12 (bacteria)
#6: DNA chain Template DNA (34-MER)


Mass: 10500.740 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli K-12 (bacteria)

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Details

Has ligand of interestN
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: RNA polymerase-sigma54-PspF(1-275) intermediate complex bound to nifH DNA (-28 to +35) containing mismatch from -11 to -8, conformation 6
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli K-12 (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 7.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 22724 / Algorithm: BACK PROJECTION / Symmetry type: POINT
RefinementHighest resolution: 7.2 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00327080
ELECTRON MICROSCOPYf_angle_d0.5537886
ELECTRON MICROSCOPYf_dihedral_angle_d14.4955854
ELECTRON MICROSCOPYf_chiral_restr0.0425075
ELECTRON MICROSCOPYf_plane_restr0.0035198

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