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- EMDB-17581: Cryo-EM structure of the c-di-GMP-bound FleQ-FleN master regulato... -

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Basic information

Entry
Database: EMDB / ID: EMD-17581
TitleCryo-EM structure of the c-di-GMP-bound FleQ-FleN master regulator complex from Pseudomonas aeruginosa
Map dataDeep EMhancer sharpened map of the c-di-GMP-bound FleQ-FleN master regulator complex of P. aeruginosa
Sample
  • Complex: C-di-GMP bound FleQ-FleN complex from Pseudomonas aeruginosa
    • Protein or peptide: Transcriptional regulator FleQ
    • Protein or peptide: Antiactivator FleN
  • Ligand: 9,9'-[(2R,3R,3aS,5S,7aR,9R,10R,10aS,12S,14aR)-3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetraoxadiphosphacyclododecine-2,9-diyl]bis(2-amino-1,9-dihydro-6H-purin-6-one)
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER
KeywordsBiofilm formation / c-di-GMP signaling / second messengers / bacterial secretion / gene regulation / bEBPs
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 / negative regulation of extracellular matrix assembly / positive regulation of cell-substrate adhesion / DNA-binding transcription repressor activity / DNA-binding transcription activator 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 / negative regulation of extracellular matrix assembly / positive regulation of cell-substrate adhesion / DNA-binding transcription repressor activity / DNA-binding transcription activator 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. / Sigma-54 interaction domain, ATP-binding site 2 ...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
Transcriptional regulator FleQ / Antiactivator FleN
Similarity search - Component
Biological speciesPseudomonas aeruginosa PAO1 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsTorres-Sanchez LT / Krasteva PV
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)BioMatrix 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
DepositionJun 8, 2023-
Header (metadata) releaseDec 13, 2023-
Map releaseDec 13, 2023-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17581.png

Downloads & links

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Map

FileDownload / File: emd_17581.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDeep EMhancer sharpened map of the c-di-GMP-bound FleQ-FleN master regulator complex of P. aeruginosa
Voxel sizeX=Y=Z: 1.419 Å
Density
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.001647067 - 1.9891975
Average (Standard dev.)0.0011743561 (±0.02463723)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 283.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened map of the c-di-GMP-bound FleQ-FleN master regulator...

Fileemd_17581_additional_1.map
AnnotationUnsharpened map of the c-di-GMP-bound FleQ-FleN master regulator complex of P. aeruginosa
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map of the c-di-GMP-bound FleQ-FleN master regulator complex...

Fileemd_17581_half_map_1.map
AnnotationHalf-map of the c-di-GMP-bound FleQ-FleN master regulator complex of P. aeruginosa
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map of the c-di-GMP-bound FleQ-FleN master regulator complex...

Fileemd_17581_half_map_2.map
AnnotationHalf-map of the c-di-GMP-bound FleQ-FleN master regulator complex of P. aeruginosa
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : C-di-GMP bound FleQ-FleN complex from Pseudomonas aeruginosa

EntireName: C-di-GMP bound FleQ-FleN complex from Pseudomonas aeruginosa
Components
  • Complex: C-di-GMP bound FleQ-FleN complex from Pseudomonas aeruginosa
    • Protein or peptide: Transcriptional regulator FleQ
    • Protein or peptide: Antiactivator FleN
  • Ligand: 9,9'-[(2R,3R,3aS,5S,7aR,9R,10R,10aS,12S,14aR)-3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetraoxadiphosphacyclododecine-2,9-diyl]bis(2-amino-1,9-dihydro-6H-purin-6-one)
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER

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Supramolecule #1: C-di-GMP bound FleQ-FleN complex from Pseudomonas aeruginosa

SupramoleculeName: C-di-GMP bound FleQ-FleN complex from Pseudomonas aeruginosa
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: C-di-GMP bound complex between FleQ REC and AAA+ domains and FleN D48A mutant from Pseudomonas aeruginosa
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Molecular weightTheoretical: 233 KDa

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Macromolecule #1: Transcriptional regulator FleQ

MacromoleculeName: Transcriptional regulator FleQ / type: protein_or_peptide / ID: 1
Details: FleQ-receiver and AAA+ domains of Pseudomonas aeruginosa FleQ
Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Molecular weightTheoretical: 44.884316 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSWRETKLL LIDDNLDRSR DLAVILNFLG EDQLTCNSED WREVAAGLSN SREALCVLLG SVESKGGAVE LLKQLASWDE YLPILLIGE PAPADWPEEL RRRVLASLEM PPSYNKLLDS LHRAQVYREM YDQARERGRS REPNLFRSLV GTSRAIQQVR Q MMQQVADT ...String:
MGSWRETKLL LIDDNLDRSR DLAVILNFLG EDQLTCNSED WREVAAGLSN SREALCVLLG SVESKGGAVE LLKQLASWDE YLPILLIGE PAPADWPEEL RRRVLASLEM PPSYNKLLDS LHRAQVYREM YDQARERGRS REPNLFRSLV GTSRAIQQVR Q MMQQVADT DASVLILGES GTGKEVVARN LHYHSKRREG PFVPVNCGAI PAELLESELF GHEKGAFTGA ITSRAGRFEL AN GGTLFLD EIGDMPLPMQ VKLLRVLQER TFERVGSNKT QNVDVRIIAA THKNLEKMIE DGTFREDLYY RLNVFPIEMA PLR ERVEDI ALLLNELISR MEHEKRGSIR FNSAAIMSLC RHDWPGNVRE LANLVERLAI MHPYGVIGVG ELPKKFRHVD

UniProtKB: Transcriptional regulator FleQ

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Macromolecule #2: Antiactivator FleN

MacromoleculeName: Antiactivator FleN / type: protein_or_peptide / ID: 2 / Details: Pseudomonas aeruginosa FleN D48A mutant / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Molecular weightTheoretical: 30.327141 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GPMGSKQMGS MHPVQVIAVT GGKGGVGKTN VSVNLALALA DLGRRVMLLD AALGLANVDV LLGLTPKRTL ADVIEGRCEL RDVLLLGPG GVRIVPAASG TQSMVHLSPM QHAGLIQAFS DISDNLDVLV VDTAAGIGDS VVSFVRAAQE VLLVVCDEPT S ITDAYALI ...String:
GPMGSKQMGS MHPVQVIAVT GGKGGVGKTN VSVNLALALA DLGRRVMLLD AALGLANVDV LLGLTPKRTL ADVIEGRCEL RDVLLLGPG GVRIVPAASG TQSMVHLSPM QHAGLIQAFS DISDNLDVLV VDTAAGIGDS VVSFVRAAQE VLLVVCDEPT S ITDAYALI KLLNRDHGMT RFRVLANMAH SPQEGRNLFA KLTKVTDRFL DVALQYVGVI PYDESVRKAV QKQRAVYEAF PR SKASLAF KAVAQKVDSW PLPANPRGHL EFFVERLVQH PATGSAV

UniProtKB: Antiactivator FleN

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Macromolecule #3: 9,9'-[(2R,3R,3aS,5S,7aR,9R,10R,10aS,12S,14aR)-3,5,10,12-tetrahydr...

MacromoleculeName: 9,9'-[(2R,3R,3aS,5S,7aR,9R,10R,10aS,12S,14aR)-3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetraoxadiphosphacyclododecine-2,9-diyl]bis(2-amino-1,9-dihydro-6H-purin-6-one)
type: ligand / ID: 3 / Number of copies: 2 / Formula: C2E
Molecular weightTheoretical: 690.411 Da
Chemical component information

ChemComp-C2E:
9,9'-[(2R,3R,3aS,5S,7aR,9R,10R,10aS,12S,14aR)-3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetraoxadiphosphacyclododecine-2,9-diyl]bis(2-amino-1,9-dihydro-6H-purin-6-one)

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Macromolecule #4: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER

MacromoleculeName: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 2 / Formula: ACP
Molecular weightTheoretical: 505.208 Da
Chemical component information

ChemComp-ACP:
PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER / AMP-PCP, energy-carrying molecule analogue*YM

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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
Details: 20 mM HEPES pH 8.0, 250mM NaCl, 2mM MgCl2, and 2% glycerol, 4 uM c-di-GMP, 200 uM ACP
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: triple deposition.
DetailsFleN-D48A and FleQ (REC-AAA+) coexpression from the pProEx-Htb and pRSFDuet1 vectors. Purified via the HRV3c- cleavable N-terminal hexahistidine tag on FleN

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 14640 / Average electron dose: 50.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
In silico model: Ab initio cryosparc Crystal structure fitting for handedness determination
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: OTHER / Software - Name: cryoSPARC
Details: Due to flexibility, preferential orientation and partial occupancy for one of the subunits, the data was downsampled and the resolution used for refinement was cut to 3.3 angstrom after Deep EMhancer sharpening.
Number images used: 1283732
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC
Details: 2 045 780 particles total Class I : 1 283 732 particles (63%)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7ejw


Chain - Source name: Other / Chain - Initial model type: experimental model
Details: the model was interpreted by modular fitting of partial crystal structures and refined against the electron density
DetailsCrystal structures fitting Refined in Phenix, coot and Namdinator
RefinementSpace: REAL
Output model

PDB-8pb9:
Cryo-EM structure of the c-di-GMP-bound FleQ-FleN master regulator complex from Pseudomonas aeruginosa

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