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- EMDB-17445: Cryo-EM structure of the c-di-GMP-free FleQ-FleN master regulator... -

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Basic information

Entry
Database: EMDB / ID: EMD-17445
TitleCryo-EM structure of the c-di-GMP-free FleQ-FleN master regulator complex of P. aeruginosa
Map dataSharpened cryo-EM density map of the c-di-GMP-free FleQ-FleN master regulator complex
Sample
  • Complex: C-di-GMP-free FleQ-FleN complex
    • Protein or peptide: Antiactivator FleN
    • Protein or peptide: Transcriptional regulator FleQ
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER
KeywordsPseudomonas aeruginosa / biofilm / c-di-GMP / transcription regulation / GENE 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 / 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: 2.7 Å
AuthorsTorres-Sanchez L / Krasteva PV
Funding supportEuropean Union, 1 items
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, 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_17445.png

Downloads & links

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Map

FileDownload / File: emd_17445.map.gz / Format: CCP4 / Size: 669.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened cryo-EM density map of the c-di-GMP-free FleQ-FleN master regulator complex
Voxel sizeX=Y=Z: 0.66 Å
Density
Contour LevelBy AUTHOR: 0.028
Minimum - Maximum-0.0017903589 - 1.7706428
Average (Standard dev.)0.0006304255 (±0.017879432)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions560560560
Spacing560560560
CellA=B=C: 369.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened cryo-EM density map of the c-di-GMP-free FleQ-FleN...

Fileemd_17445_additional_1.map
AnnotationUnsharpened cryo-EM density map of the c-di-GMP-free FleQ-FleN master regulator complex
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-free FleQ-FleN master regulator complex

Fileemd_17445_half_map_1.map
AnnotationHalf-map of the c-di-GMP-free FleQ-FleN master regulator complex
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-free FleQ-FleN master regulator complex

Fileemd_17445_half_map_2.map
AnnotationHalf-map of the c-di-GMP-free FleQ-FleN master regulator complex
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-free FleQ-FleN complex

EntireName: C-di-GMP-free FleQ-FleN complex
Components
  • Complex: C-di-GMP-free FleQ-FleN complex
    • Protein or peptide: Antiactivator FleN
    • Protein or peptide: Transcriptional regulator FleQ
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER

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Supramolecule #1: C-di-GMP-free FleQ-FleN complex

SupramoleculeName: C-di-GMP-free FleQ-FleN complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Full-length FleQ co-purified with His-tagged FleN D48A
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Molecular weightTheoretical: 288 KDa

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Macromolecule #1: Antiactivator FleN

MacromoleculeName: Antiactivator FleN / type: protein_or_peptide / ID: 1 / Details: P. aeruginosa FleN D48A / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria) / Strain: PAO1
Molecular weightTheoretical: 33.236332 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSYYHHHHHH DYDIPTTLEV LFQGPMGSKQ MGSMHPVQVI AVTGGKGGVG KTNVSVNLAL ALADLGRRVM LLDAALGLAN VDVLLGLTP KRTLADVIEG RCELRDVLLL GPGGVRIVPA ASGTQSMVHL SPMQHAGLIQ AFSDISDNLD VLVVDTAAGI G DSVVSFVR ...String:
MSYYHHHHHH DYDIPTTLEV LFQGPMGSKQ MGSMHPVQVI AVTGGKGGVG KTNVSVNLAL ALADLGRRVM LLDAALGLAN VDVLLGLTP KRTLADVIEG RCELRDVLLL GPGGVRIVPA ASGTQSMVHL SPMQHAGLIQ AFSDISDNLD VLVVDTAAGI G DSVVSFVR AAQEVLLVVC DEPTSITDAY ALIKLLNRDH GMTRFRVLAN MAHSPQEGRN LFAKLTKVTD RFLDVALQYV GV IPYDESV RKAVQKQRAV YEAFPRSKAS LAFKAVAQKV DSWPLPANPR GHLEFFVERL VQHPATGSAV

UniProtKB: Antiactivator FleN

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Macromolecule #2: Transcriptional regulator FleQ

MacromoleculeName: Transcriptional regulator FleQ / type: protein_or_peptide / ID: 2
Details: ...Details: MGSWRETKLLLIDDNLDRSRDLAVILNFLGEDQLTCNSEDWREVAAGLSNSREALCVLLGSVESKGGAVELLKQLASWDEYLPILLIGEPAPADWPEELRRRVLASLEMPPSYNKLLDSLHRAQVYREMYDQARERGRSREPNLFRSLVGTSRAIQQVRQMMQQVADTDASVLILGESGTGKEVVARNLHYHSKRREGPFVPVNCGAIPAELLESELFGHEKGAFTGAITSRAGRFELANGGTLFLDEIGDMPLPMQVKLLRVLQERTFERVGSNKTQNVDVRIIAATHKNLEKMIEDGTFREDLYYRLNVFPIEMAPLRERVEDIALLLNELISRMEHEKRGSIRFNSAAIMSLCRHDWPGNVRELANLVERLAIMHPYGVIGVGELPKKFRHVDDEDEQLASSLREELEERAAINAGLPGMDAPAMLPAEGLDLKDYLANLEQGLIQQALDDAGGVVARAAERLRIRRTTLVEKMRKYGMSRRDDDLSDD
Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Molecular weightTheoretical: 55.494125 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 DEDE QLASS LREELEERAA INAGLPGMDA PAMLPAEGLD LKDYLANLEQ GLIQQALDDA GGVVARAAER LRIRRTTLVE KMRKY GMSR RDDDLSDD

UniProtKB: Transcriptional regulator FleQ

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration2 mg/mL
BufferpH: 8
Details: 20 mM HEPES pH 8.0, 250mM NaCl, 2mM MgCl2, and 2% glycerol
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: 4 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 21233 / Average electron dose: 51.5 e/Å2 / Details: 17099 micrographs retained for processing
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: 2.5 µm / Nominal defocus min: 0.3 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4124200 / Details: 404 228 particles retained for refinement
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 404228
Initial angle assignmentType: OTHER / Details: cryoSPARC Ab-initio
Final angle assignmentType: OTHER
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC
Details: 90 % of aligned particles in one of two 3D classes.
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

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

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