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- PDB-8p53: Cryo-EM structure of the c-di-GMP-free FleQ-FleN master regulator... -

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Basic information

Entry
Database: PDB / ID: 8p53
TitleCryo-EM structure of the c-di-GMP-free FleQ-FleN master regulator complex of P. aeruginosa
Components
  • Antiactivator FleN
  • Transcriptional regulator FleQ
KeywordsGENE REGULATION / Pseudomonas aeruginosa / biofilm / c-di-GMP / transcription regulation
Function / homology
Function and homology information


positive regulation of cilium-dependent cell motility / regulation of bacterial-type flagellum-dependent cell motility / negative regulation of cell division / cyclic-di-GMP binding / positive regulation of cell-substrate adhesion / negative regulation of extracellular matrix assembly / DNA-binding transcription activator activity / DNA-binding transcription repressor activity / cis-regulatory region sequence-specific DNA binding / protein-DNA complex ...positive regulation of cilium-dependent cell motility / regulation of bacterial-type flagellum-dependent cell motility / negative regulation of cell division / cyclic-di-GMP binding / positive regulation of cell-substrate adhesion / negative regulation of extracellular matrix assembly / DNA-binding transcription activator activity / DNA-binding transcription repressor activity / cis-regulatory region sequence-specific DNA binding / protein-DNA complex / cytoplasmic side of plasma membrane / transcription cis-regulatory region binding / DNA-templated transcription / regulation of DNA-templated transcription / ATP hydrolysis activity / ATP binding / cytosol
Similarity search - Function
Flagellum site-determining protein FlhG / Flagellar regulatory FleQ / Flagellar regulatory protein FleQ / Flagellum site-determining protein YlxH/ Fe-S cluster assembling factor NBP35 / NUBPL iron-transfer P-loop NTPase / ATP binding protein MinD/FleN / : / Sigma-54 interaction domain ATP-binding region A signature. / Sigma-54 interaction domain, conserved site / Sigma-54 interaction domain C-terminal part signature. ...Flagellum site-determining protein FlhG / Flagellar regulatory FleQ / Flagellar regulatory protein FleQ / Flagellum site-determining protein YlxH/ Fe-S cluster assembling factor NBP35 / NUBPL iron-transfer P-loop NTPase / ATP binding protein MinD/FleN / : / Sigma-54 interaction domain ATP-binding region A signature. / 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, ATP-binding site 1 / 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 / CheY-like superfamily / 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
PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER / Transcriptional regulator FleQ / Antiactivator FleN
Similarity search - Component
Biological speciesPseudomonas aeruginosa PAO1 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsTorres-Sanchez, L. / Krasteva, P.V.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)757507European Union
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Structures of the FleQ-FleN master regulators reveal large-scale conformational switching in motility and biofilm control.
Authors: Lucía Torres-Sánchez / Thibault Géry Sana / Marion Decossas / Yaser Hashem / Petya Violinova Krasteva /
Abstract: can cause a wide array of chronic and acute infections associated with its ability to rapidly switch between planktonic, biofilm, and dispersed lifestyles, each with a specific arsenal for bacterial ... can cause a wide array of chronic and acute infections associated with its ability to rapidly switch between planktonic, biofilm, and dispersed lifestyles, each with a specific arsenal for bacterial survival and virulence. At the cellular level, many of the physiological transitions are orchestrated by the intracellular second messenger c-di-GMP and its receptor-effector FleQ. A bacterial enhancer binding protein, FleQ acts as a master regulator of both flagellar motility and adherence factor secretion and uses remarkably different transcription activation mechanisms depending on its dinucleotide loading state, adenosine triphosphatase (ATPase) activity, interactions with polymerase sigma (σ) factors, and complexation with a second ATPase, FleN. How the FleQ-FleN tandem can exert diverse effects through recognition of a conserved FleQ binding consensus has remained enigmatic. Here, we provide cryogenic electron microscopy (cryo-EM) structures of both c-di-GMP-bound and c-di-GMP-free FleQ-FleN complexes which deepen our understanding of the proteins' (di)nucleotide-dependent conformational switching and fine-tuned roles in gene expression regulation.
History
DepositionMay 23, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 13, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Antiactivator FleN
B: Antiactivator FleN
C: Transcriptional regulator FleQ
D: Transcriptional regulator FleQ
E: Transcriptional regulator FleQ
F: Transcriptional regulator FleQ
hetero molecules


Theoretical massNumber of molelcules
Total (without water)289,50810
Polymers288,4496
Non-polymers1,0594
Water00
1


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

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Components

#1: Protein Antiactivator FleN


Mass: 33236.332 Da / Num. of mol.: 2 / Mutation: FleN D48A
Source method: isolated from a genetically manipulated source
Details: P. aeruginosa FleN D48A / Source: (gene. exp.) Pseudomonas aeruginosa PAO1 (bacteria) / Strain: PAO1 / Gene: fleN, PA1454 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: G3XD64
#2: Protein
Transcriptional regulator FleQ


Mass: 55494.125 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: ...Details: MGSWRETKLLLIDDNLDRSRDLAVILNFLGEDQLTCNSEDWREVAAGLSNSREALCVLLGSVESKGGAVELLKQLASWDEYLPILLIGEPAPADWPEELRRRVLASLEMPPSYNKLLDSLHRAQVYREMYDQARERGRSREPNLFRSLVGTSRAIQQVRQMMQQVADTDASVLILGESGTGKEVVARNLHYHSKRREGPFVPVNCGAIPAELLESELFGHEKGAFTGAITSRAGRFELANGGTLFLDEIGDMPLPMQVKLLRVLQERTFERVGSNKTQNVDVRIIAATHKNLEKMIEDGTFREDLYYRLNVFPIEMAPLRERVEDIALLLNELISRMEHEKRGSIRFNSAAIMSLCRHDWPGNVRELANLVERLAIMHPYGVIGVGELPKKFRHVDDEDEQLASSLREELEERAAINAGLPGMDAPAMLPAEGLDLKDYLANLEQGLIQQALDDAGGVVARAAERLRIRRTTLVEKMRKYGMSRRDDDLSDD
Source: (gene. exp.) Pseudomonas aeruginosa PAO1 (bacteria) / Gene: fleQ, PA1097 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: G3XCV0
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-ACP / PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER / ADENOSINE-5'-[BETA, GAMMA-METHYLENE]TRIPHOSPHATE


Mass: 505.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C11H18N5O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PCP, energy-carrying molecule analogue*YM
Has ligand of interestY

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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: C-di-GMP-free FleQ-FleN complex / Type: COMPLEX
Details: Full-length FleQ co-purified with His-tagged FleN D48A
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.288 MDa / Experimental value: NO
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Details: 20 mM HEPES pH 8.0, 250mM NaCl, 2mM MgCl2, and 2% glycerol
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 4 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 51.5 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 21233 / Details: 17099 micrographs retained for processing
EM imaging opticsEnergyfilter name: GIF Quantum LS
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
4GctfCTF correction
7Cootmodel fitting
8UCSF ChimeraXmodel fitting
12cryoSPARCclassification
13cryoSPARC3D reconstruction
14PHENIXmodel refinement
CTF correctionDetails: Gctf software / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4124200 / Details: 404 228 particles retained for refinement
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 404228 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00316583
ELECTRON MICROSCOPYf_angle_d0.61522442
ELECTRON MICROSCOPYf_dihedral_angle_d4.7962319
ELECTRON MICROSCOPYf_chiral_restr0.0432554
ELECTRON MICROSCOPYf_plane_restr0.0042931

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