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- PDB-9wsf: Crystal structure of Sigma28/FlgM complex from Pseudomonas aerugi... -

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Basic information

Entry
Database: PDB / ID: 9wsf
TitleCrystal structure of Sigma28/FlgM complex from Pseudomonas aeruginosa at 1.95 Angstrom resolution
Components
  • Negative regulator of flagellin synthesis
  • RNA polymerase sigma factor FliA
KeywordsTRANSCRIPTION / P. aeruginosa / FliA/FlgM / Flagella
Function / homology
Function and homology information


bacterial-type flagellum organization / positive regulation of cell motility / sigma factor activity / DNA-templated transcription initiation / DNA-directed RNA polymerase activity / negative regulation of DNA-templated transcription / regulation of DNA-templated transcription / DNA binding / cytoplasm
Similarity search - Function
Anti-sigma-28 factor, FlgM / Anti-sigma-28 factor FlgM, C-terminal / Anti-sigma-28 factor FlgM superfamily / Anti-sigma-28 factor, FlgM / 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 ...Anti-sigma-28 factor, FlgM / Anti-sigma-28 factor FlgM, C-terminal / Anti-sigma-28 factor FlgM superfamily / Anti-sigma-28 factor, FlgM / 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
Similarity search - Domain/homology
Negative regulator of flagellin synthesis / RNA polymerase sigma factor FliA
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.95 Å
AuthorsNln, S. / Kumar, V. / Sahoo, P.K. / Jain, D.
Funding support India, 1items
OrganizationGrant numberCountry
Science and Engineering Research Board (SERB) 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA polymerase sigma factor FliA
B: Negative regulator of flagellin synthesis


Theoretical massNumber of molelcules
Total (without water)39,8882
Polymers39,8882
Non-polymers00
Water3,153175
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3390 Å2
ΔGint-20 kcal/mol
Surface area14430 Å2
MethodPISA
Unit cell
Length a, b, c (Å)56.028, 139.890, 74.308
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221
Space group name HallC2c2
Symmetry operation#1: x,y,z
#2: x,-y,-z
#3: -x,y,-z+1/2
#4: -x,-y,z+1/2
#5: x+1/2,y+1/2,z
#6: x+1/2,-y+1/2,-z
#7: -x+1/2,y+1/2,-z+1/2
#8: -x+1/2,-y+1/2,z+1/2
Components on special symmetry positions
IDModelComponents
11A-318-

HOH

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Components

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


Mass: 28213.920 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
#2: Protein Negative regulator of flagellin synthesis / Anti-sigma-28 factor


Mass: 11673.864 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria)
Gene: flgM, ALP65_00594, CAZ10_21890, DT376_16730, GNQ48_13085, GUL26_03340, IPC1295_29415, PAERUG_P19_London_7_VIM_2_05_10_00578
Production host: Escherichia coli (E. coli) / References: UniProt: G3XCL4
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 175 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.41 Å3/Da / Density % sol: 48.9 % / Description: Thin sharp plates
Crystal growTemperature: 281.15 K / Method: vapor diffusion, hanging drop / pH: 5
Details: 15% PEG 8000, 0.1 M sodium cacodylate pH 5.0, 0.2 M magnesium acetate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: BM30A / Wavelength: 0.965459 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Nov 24, 2023
RadiationMonochromator: M / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.965459 Å / Relative weight: 1
ReflectionResolution: 1.95→69.94 Å / Num. obs: 20852 / % possible obs: 96.6 % / Redundancy: 3.5 % / Biso Wilson estimate: 35.5 Å2 / CC1/2: 0.994 / Rmerge(I) obs: 0.059 / Rpim(I) all: 0.034 / Rrim(I) all: 0.069 / Χ2: 0.69 / Net I/σ(I): 7.7
Reflection shellResolution: 1.95→2 Å / Redundancy: 3.3 % / Rmerge(I) obs: 0.396 / Mean I/σ(I) obs: 2.6 / Num. unique obs: 1489 / CC1/2: 0.803 / Rpim(I) all: 0.233 / Rrim(I) all: 0.464 / Χ2: 0.82 / % possible all: 98.7

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Processing

Software
NameVersionClassification
iMOSFLMdata reduction
Aimlessdata scaling
PHENIXphasing
Cootmodel building
PHENIXv1.17.1-3660refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: AlphaFold

Resolution: 1.95→52.01 Å / SU ML: 0.215 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 23.7095
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.226 1042 5.01 %
Rwork0.1773 19776 -
obs0.1797 20818 95.84 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 58.85 Å2
Refinement stepCycle: LAST / Resolution: 1.95→52.01 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2118 0 0 175 2293
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00682157
X-RAY DIFFRACTIONf_angle_d0.82312910
X-RAY DIFFRACTIONf_chiral_restr0.0549326
X-RAY DIFFRACTIONf_plane_restr0.0047385
X-RAY DIFFRACTIONf_dihedral_angle_d6.0967303
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.95-2.050.29391610.23752821X-RAY DIFFRACTION98.22
2.05-2.180.25191450.20312829X-RAY DIFFRACTION96.81
2.18-2.350.23151570.19092808X-RAY DIFFRACTION96.67
2.35-2.590.23921330.17252840X-RAY DIFFRACTION96.24
2.59-2.960.22651550.18022774X-RAY DIFFRACTION95.47
2.96-3.730.21381360.17312862X-RAY DIFFRACTION95.42
3.73-52.010.21771550.16742842X-RAY DIFFRACTION92.33
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
14.36403231098-0.70743797271-0.8380152554262.690286311462.704587383046.27188384097-0.1389690514751.013717569150.0202734931525-1.022157392810.127385608956-0.340959208706-0.02565165379740.203207941889-0.002957054107270.709056363539-0.06701819448820.1712012801960.5315496667070.01589016099720.39139966317614.70010666899.22434082571-15.4265962863
22.10854290505-0.670419397025-2.078289788813.305989583711.931883488236.89197784176-0.2095851381660.210941584849-0.0810580471359-0.444766619815-0.1681464687790.09493169696270.00899044060991-0.1724775152720.3466055436160.244589486097-0.0174972953010.03252058166410.2605278151820.0318854956150.3317117634947.4594751096616.99815626256.21383978008
33.29716841387-2.856593744232.005391255672.92232724663-1.962645510314.80110924818-0.467244184256-0.3313096803470.2882616117850.7011592367040.3728147509680.0986039071805-0.523357958876-0.6502279929420.03332285790480.2897988018660.0602922263038-0.03212214732750.3265392258180.02248409063160.4281583354984.090147027738.0448792227.09355205637
44.89742921661-0.933545494748-3.41607890523.039989889591.252138321796.7814782988-0.120056247102-0.136850721062-0.1084498035970.136982755181-0.0470975134072-0.08467890705120.09801647363230.2838308780070.1073777284310.164492609828-0.003004114971920.01224284415750.2044779730130.07459759094090.29316247610916.185900109319.99055277888.19699980592
53.32486804821-0.3462158970221.335079347985.104255278151.585660127494.542933639830.6146366448852.40860757968-1.76429583828-0.5047984649721.03367061073-0.9536817852232.638016915241.15933716083-1.521423441571.489821613410.1065340918260.2813418646591.10141311542-0.1788822626181.0688295740121.68070024521.18665223649-16.8499428783
62.82706687555-2.097190071020.8112532302963.34175749189-0.4312757719990.2420274098310.05384539775161.70167940392-0.933890091179-0.7200124934840.727039420716-1.000781164492.035589013250.872936704992-1.520368533751.015318693450.00443876435960.1234937436630.735461460778-0.2776124630440.60944540995526.17077716446.55526603216-8.55679154105
76.55837180791-0.5505177917831.382499915614.943834927253.934199692865.56518909239-0.2972944968790.261788443812-0.101710949479-0.2463425943980.408077620787-2.1426505839-0.2360419379551.75260237375-0.2866719173160.2774370017990.0577781938170.03412700310120.6181378799010.1094533710170.6194054004632.908188917319.02474754061.74940760458
82.767827944880.0631699865508-0.4627188404475.24903226002-0.2723227587717.24285603078-0.04604220351850.6187168669650.487219693988-0.4798826080520.1512357743620.256924785533-0.0671706483029-0.0771266798941-0.1095454449650.242674657668-0.00529514975631-0.02730297376640.2765066954060.08445122470050.3137831693715.724263473926.58272436850.29626133034
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 14 through 63 )
2X-RAY DIFFRACTION2chain 'A' and (resid 64 through 155 )
3X-RAY DIFFRACTION3chain 'A' and (resid 156 through 193 )
4X-RAY DIFFRACTION4chain 'A' and (resid 194 through 247 )
5X-RAY DIFFRACTION5chain 'B' and (resid 62 through 66 )
6X-RAY DIFFRACTION6chain 'B' and (resid 67 through 73 )
7X-RAY DIFFRACTION7chain 'B' and (resid 74 through 86 )
8X-RAY DIFFRACTION8chain 'B' and (resid 87 through 107 )

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