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Yorodumi- EMDB-17445: Cryo-EM structure of the c-di-GMP-free FleQ-FleN master regulator... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-17445 | |||||||||
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Title | Cryo-EM structure of the c-di-GMP-free FleQ-FleN master regulator complex of P. aeruginosa | |||||||||
Map data | Sharpened cryo-EM density map of the c-di-GMP-free FleQ-FleN master regulator complex | |||||||||
Sample |
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Keywords | Pseudomonas aeruginosa / biofilm / c-di-GMP / transcription regulation / GENE REGULATION | |||||||||
Function / homology | Function and homology information negative regulation of extracellular matrix assembly / 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 / DNA-binding transcription repressor activity / DNA-binding transcription activator activity / cis-regulatory region sequence-specific DNA binding / protein-DNA complex ...negative regulation of extracellular matrix assembly / 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 / 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 | |||||||||
Biological species | Pseudomonas aeruginosa PAO1 (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.7 Å | |||||||||
Authors | Torres-Sanchez L / Krasteva PV | |||||||||
Funding support | European Union, 1 items
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_17445.map.gz | 587.9 MB | EMDB map data format | |
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Header (meta data) | emd-17445-v30.xml emd-17445.xml | 22 KB 22 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_17445_fsc.xml | 18.5 KB | Display | FSC data file |
Images | emd_17445.png | 93.9 KB | ||
Filedesc metadata | emd-17445.cif.gz | 6.8 KB | ||
Others | emd_17445_additional_1.map.gz emd_17445_half_map_1.map.gz emd_17445_half_map_2.map.gz | 328.7 MB 621 MB 621 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-17445 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-17445 | HTTPS FTP |
-Validation report
Summary document | emd_17445_validation.pdf.gz | 661.2 KB | Display | EMDB validaton report |
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Full document | emd_17445_full_validation.pdf.gz | 660.8 KB | Display | |
Data in XML | emd_17445_validation.xml.gz | 28.4 KB | Display | |
Data in CIF | emd_17445_validation.cif.gz | 37.2 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-17445 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-17445 | HTTPS FTP |
-Related structure data
Related structure data | 8p53MC 8pb9C M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_17445.map.gz / Format: CCP4 / Size: 669.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | Sharpened cryo-EM density map of the c-di-GMP-free FleQ-FleN master regulator complex | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.66 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: Unsharpened cryo-EM density map of the c-di-GMP-free FleQ-FleN...
File | emd_17445_additional_1.map | ||||||||||||
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Annotation | Unsharpened cryo-EM density map of the c-di-GMP-free FleQ-FleN master regulator complex | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half-map of the c-di-GMP-free FleQ-FleN master regulator complex
File | emd_17445_half_map_1.map | ||||||||||||
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Annotation | Half-map of the c-di-GMP-free FleQ-FleN master regulator complex | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half-map of the c-di-GMP-free FleQ-FleN master regulator complex
File | emd_17445_half_map_2.map | ||||||||||||
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Annotation | Half-map of the c-di-GMP-free FleQ-FleN master regulator complex | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : C-di-GMP-free FleQ-FleN complex
Entire | Name: C-di-GMP-free FleQ-FleN complex |
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Components |
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-Supramolecule #1: C-di-GMP-free FleQ-FleN complex
Supramolecule | Name: 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 |
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Source (natural) | Organism: Pseudomonas aeruginosa PAO1 (bacteria) |
Molecular weight | Theoretical: 288 KDa |
-Macromolecule #1: Antiactivator FleN
Macromolecule | Name: Antiactivator FleN / type: protein_or_peptide / ID: 1 / Details: P. aeruginosa FleN D48A / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Pseudomonas aeruginosa PAO1 (bacteria) / Strain: PAO1 |
Molecular weight | Theoretical: 33.236332 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: 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 |
-Macromolecule #2: Transcriptional regulator FleQ
Macromolecule | Name: Transcriptional regulator FleQ / type: protein_or_peptide / ID: 2 Details: ...Details: MGSWRETKLLLIDDNLDRSRDLAVILNFLGEDQLTCNSEDWREVAAGLSNSREALCVLLGSVESKGGAVELLKQLASWDEYLPILLIGEPAPADWPEELRRRVLASLEMPPSYNKLLDSLHRAQVYREMYDQARERGRSREPNLFRSLVGTSRAIQQVRQMMQQVADTDASVLILGESGTGKEVVARNLHYHSKRREGPFVPVNCGAIPAELLESELFGHEKGAFTGAITSRAGRFELANGGTLFLDEIGDMPLPMQVKLLRVLQERTFERVGSNKTQNVDVRIIAATHKNLEKMIEDGTFREDLYYRLNVFPIEMAPLRERVEDIALLLNELISRMEHEKRGSIRFNSAAIMSLCRHDWPGNVRELANLVERLAIMHPYGVIGVGELPKKFRHVDDEDEQLASSLREELEERAAINAGLPGMDAPAMLPAEGLDLKDYLANLEQGLIQQALDDAGGVVARAAERLRIRRTTLVEKMRKYGMSRRDDDLSDD Number of copies: 4 / Enantiomer: LEVO |
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Source (natural) | Organism: Pseudomonas aeruginosa PAO1 (bacteria) |
Molecular weight | Theoretical: 55.494125 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: 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 |
-Macromolecule #3: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #4: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER
Macromolecule | Name: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 2 / Formula: ACP |
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Molecular weight | Theoretical: 505.208 Da |
Chemical component information | ChemComp-ACP: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 2 mg/mL |
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Buffer | pH: 8 Details: 20 mM HEPES pH 8.0, 250mM NaCl, 2mM MgCl2, and 2% glycerol |
Grid | Model: 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 |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 4 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Quantum LS |
Image recording | Film 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 beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.3 µm |
Sample stage | Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: AB INITIO MODEL |
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Output model | PDB-8p53: |