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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-3877 | |||||||||||||||
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Title | Inner membrane Bcs complex (E. coli) | |||||||||||||||
![]() | An inner membrane subcomplex of the E. coli cellulose secretion system (Bcs). The complex was purified from solubilised membranes of Bl21(DE3) cells over-expressing the BcsRQAB subunits. | |||||||||||||||
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Biological species | ![]() ![]() | |||||||||||||||
Method | single particle reconstruction / negative staining / Resolution: 21.1 Å | |||||||||||||||
![]() | Krasteva PV / Fronzes R | |||||||||||||||
Funding support | ![]()
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![]() | ![]() Title: Insights into the structure and assembly of a bacterial cellulose secretion system. Authors: Petya Violinova Krasteva / Joaquin Bernal-Bayard / Laetitia Travier / Fernando Ariel Martin / Pierre-Alexandre Kaminski / Gouzel Karimova / Rémi Fronzes / Jean-Marc Ghigo / ![]() ![]() Abstract: Secreted exopolysaccharides present important determinants for bacterial biofilm formation, survival, and virulence. Cellulose secretion typically requires the concerted action of a c-di-GMP- ...Secreted exopolysaccharides present important determinants for bacterial biofilm formation, survival, and virulence. Cellulose secretion typically requires the concerted action of a c-di-GMP-responsive inner membrane synthase (BcsA), an accessory membrane-anchored protein (BcsB), and several additional Bcs components. Although the BcsAB catalytic duo has been studied in great detail, its interplay with co-expressed subunits remains enigmatic. Here we show that E. coli Bcs proteins partake in a complex protein interaction network. Electron microscopy reveals a stable, megadalton-sized macromolecular assembly, which encompasses most of the inner membrane and cytosolic Bcs components and features a previously unobserved asymmetric architecture. Heterologous reconstitution and mutational analyses point toward a structure-function model, where accessory proteins regulate secretion by affecting both the assembly and stability of the system. Altogether, these results lay the foundation for more comprehensive models of synthase-dependent exopolysaccharide secretion in biofilms and add a sophisticated secretory nanomachine to the diverse bacterial arsenal for virulence and adaptation. | |||||||||||||||
History |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 8.9 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 13.3 KB 13.3 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 6.3 KB | Display | ![]() |
Images | ![]() | 31.2 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 232.3 KB | Display | ![]() |
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Full document | ![]() | 231.4 KB | Display | |
Data in XML | ![]() | 9 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | An inner membrane subcomplex of the E. coli cellulose secretion system (Bcs). The complex was purified from solubilised membranes of Bl21(DE3) cells over-expressing the BcsRQAB subunits. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.9 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Bcs inner membrane sub-complex.
Entire | Name: Bcs inner membrane sub-complex. |
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Components |
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-Supramolecule #1: Bcs inner membrane sub-complex.
Supramolecule | Name: Bcs inner membrane sub-complex. / type: complex / ID: 1 / Parent: 0 Details: Sample purified using BcsA as bait from solubilised E. coli Bl21(DE3) cells over-expressing the BcsRQAB sub-units of cellulose-secreting E. coli 1094. |
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Source (natural) | Organism: ![]() ![]() |
Recombinant expression | Organism: ![]() ![]() |
Molecular weight | Theoretical: 1 MDa |
-Experimental details
-Structure determination
Method | negative staining |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.1 mg/mL |
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Buffer | pH: 8 Details: 20 mM HEPES 8.0 120 mM NaCl 10 glycerol 5 mM MgCl2 10 uM AppCp 2 uM c-di-GMP 0.008% LM-NPG cOmplete protease inhibitors |
Staining | Type: NEGATIVE / Material: uranyl acetate Details: 5 ul of sample were spotted on glow-discharged carbon-coated copper grids and incubated for 1 minute. Liquid was blotted off and the sample was passed through 3 drops of 10 ul 2% uranyl ...Details: 5 ul of sample were spotted on glow-discharged carbon-coated copper grids and incubated for 1 minute. Liquid was blotted off and the sample was passed through 3 drops of 10 ul 2% uranyl acetate with a 30-second incubation over the third. After that the stain was blotted off and the grid was air-dried. |
Grid | Material: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER |
Details | Samples were purified using anti-FLAG affinity gel from detergent-solubilised membranes of E. coli(DE3) cells over-expressing the BcsRQAB subunits of an E. coli 1094-derived strain (pCDF vector, IPTG-inducible overnight expression in TB, affinity tag on BcsA). Eluted samples were further purified by centrifugation over a 10-40% glycerol density gradient. |
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Electron microscopy
Microscope | FEI TECNAI F20 |
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Image recording | Film or detector model: FEI FALCON II (4k x 4k) / Number grids imaged: 2 / Average electron dose: 12.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Calibrated defocus max: 3.5 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 0.5 µm / Nominal magnification: 50000 |
Sample stage | Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Tecnai F20 / Image courtesy: FEI Company |