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- PDB-9wsm: Cryo-EM structure of Sigma28-RNAP open promoter complex from Pseu... -

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Basic information

Entry
Database: PDB / ID: 9wsm
TitleCryo-EM structure of Sigma28-RNAP open promoter complex from Pseudomonas aeruginosa
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • DNA (50-MER)
  • DNA (54-MER)
  • RNA polymerase sigma factor FliA
KeywordsTRANSCRIPTION / P. aeruginosa / Sigma 28 / RNAP / Flagella / fliC
Function / homology
Function and homology information


positive regulation of cell motility / sigma factor activity / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / protein dimerization activity ...positive regulation of cell motility / sigma factor activity / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / protein dimerization activity / regulation of DNA-templated transcription / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
RNA polymerase sigma factor FliA / RNA polymerase sigma factor, FliA/WhiG / Sigma-70 factors family signature 1. / RNA polymerase sigma-70 region 3 / Sigma-70 region 3 / RNA polymerase sigma-70 / RNA polymerase sigma-70 region 4 / Sigma-70, region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 like domain ...RNA polymerase sigma factor FliA / RNA polymerase sigma factor, FliA/WhiG / Sigma-70 factors family signature 1. / RNA polymerase sigma-70 region 3 / Sigma-70 region 3 / RNA polymerase sigma-70 / RNA polymerase sigma-70 region 4 / Sigma-70, region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 like domain / Sigma-70 region 2 / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / 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 beta subunit, bacterial-type / DNA-directed RNA polymerase, alpha subunit / 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, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / 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 Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase, RBP11-like subunit / 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
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA-directed RNA polymerase subunit alpha / RNA polymerase sigma factor FliA / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta'
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsNln, S. / Kumar, V. / Sahoo, P.K. / Kandiah, E. / Jain, D.
Funding support India, 2items
OrganizationGrant numberCountry
Science and Engineering Research Board (SERB)SPF/2022/000085 India
Department of Biotechnology (DBT, India)BT/PR36150/INF/22/214/2020 India
CitationJournal: Nucleic Acids Res / Year: 2026
Title: Structural insights into σ28-dependent transcription initiation and its regulation by anti-sigma factor in Pseudomonas aeruginosa.
Authors: Sheenu / Vineet Kumar / Pankaj Kumar Sahoo / Eaazhisai Kandiah / Deepti Jain /
Abstract: Late flagellar genes in Pseudomonas aeruginosa are transcribed by the group 3 sigma factor, FliA (σ28). σ28 drives the expression of flagellin, which assembles into the flagellar filament in this ...Late flagellar genes in Pseudomonas aeruginosa are transcribed by the group 3 sigma factor, FliA (σ28). σ28 drives the expression of flagellin, which assembles into the flagellar filament in this monoflagellated bacterium. This function is suppressed by the anti-sigma factor FlgM. Here, we present the 1.95Å resolution crystal structure of σ28-FlgM complex, along with a 3.4Å structure of σ28RNAP open promoter complex determined using single particle cryo-electron microscopy from P. aeruginosa. The σ28 adopts a compact conformation upon binding to the anti-sigma factor FlgM, which contacts all three domains of the sigma factor. This conformation is neither conducive to interactions with RNA polymerase nor the promoter DNA. The cryo-EM structure reveals base-specific interactions of σ28 domain 4 (σ4) with -35 element, flipping of -11 base of the template strand, novel interactions of template strand with domain 2 (σ2) and 3 (σ3), and partial insertion of sigma finger into the active site cleft, offering unique features of group 3 sigma interactions with promoter DNA. Perturbation of key residues affects transcription in vitro and flagellar phenotypes as well as bacterial motility in vivo. Analysis of the structural data presented here reveals new insights into transcription regulation of late flagellar genes.
History
DepositionSep 13, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 25, 2026Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data 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: DNA (54-MER)
2: DNA (50-MER)
E: DNA-directed RNA polymerase subunit omega
F: RNA polymerase sigma factor FliA
A: DNA-directed RNA polymerase subunit alpha
B: DNA-directed RNA polymerase subunit alpha
C: DNA-directed RNA polymerase subunit beta
D: DNA-directed RNA polymerase subunit beta'
hetero molecules


Theoretical massNumber of molelcules
Total (without water)450,38411
Polymers450,2298
Non-polymers1553
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA chain , 2 types, 2 molecules 12

#1: DNA chain DNA (54-MER)


Mass: 16560.641 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pseudomonas aeruginosa (bacteria)
#2: DNA chain DNA (50-MER)


Mass: 16622.664 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pseudomonas aeruginosa (bacteria)

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

#3: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 9783.876 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Pseudomonas aeruginosa (bacteria) / References: UniProt: Q9HTM1, DNA-directed RNA polymerase
#5: Protein DNA-directed RNA polymerase subunit alpha / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 36694.555 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Pseudomonas aeruginosa (bacteria) / References: UniProt: O52760, DNA-directed RNA polymerase
#6: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 151037.047 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Pseudomonas aeruginosa (bacteria) / References: UniProt: Q51561, DNA-directed RNA polymerase
#7: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 154595.000 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Pseudomonas aeruginosa (bacteria) / References: UniProt: Q9HWC9, DNA-directed RNA polymerase

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Protein , 1 types, 1 molecules F

#4: Protein RNA polymerase sigma factor FliA / RNA polymerase sigma factor for flagellar operon / Sigma F / Sigma-28


Mass: 28240.924 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: fliA, rpoF, PA1455 / Production host: Escherichia coli (E. coli) / References: UniProt: P29248

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Non-polymers , 2 types, 3 molecules

#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
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

Component
IDNameTypeEntity IDParent-IDSource
1Sigma28-RNAP open promoter complexCOMPLEX#1-#70MULTIPLE SOURCES
2DNACOMPLEX#1-#21SYNTHETIC
3sigma28COMPLEX#41RECOMBINANT
4RNA polymeraseCOMPLEX#3, #5-#71NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen 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 magnification: 105000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 41.34 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION3.1.3particle selection
4CTFFIND4.1.14CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 36636 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 100.47 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00329323
ELECTRON MICROSCOPYf_angle_d0.491540058
ELECTRON MICROSCOPYf_chiral_restr0.03974581
ELECTRON MICROSCOPYf_plane_restr0.00314889
ELECTRON MICROSCOPYf_dihedral_angle_d19.27144759

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