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- EMDB-3877: Inner membrane Bcs complex (E. coli) -

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Basic information

Entry
Database: EMDB / ID: EMD-3877
TitleInner membrane Bcs complex (E. coli)
Map dataAn 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.
Sample
  • Complex: Bcs inner membrane sub-complex.
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / negative staining / Resolution: 21.1 Å
AuthorsKrasteva PV / Fronzes R
Funding support France, 4 items
OrganizationGrant numberCountry
CNRSATIP-Avenir 2016 France
ANR/LABEXANR-10-LABX-62-IBEID to J.M.G. France
European Research CouncilMolStructTransfo, 281149 France
FRMDEQ20140329508 France
CitationJournal: Nat Commun / Year: 2017
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
DepositionSep 14, 2017-
Header (metadata) releaseDec 20, 2017-
Map releaseDec 27, 2017-
UpdateNov 6, 2019-
Current statusNov 6, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3877.png

Downloads & links

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Map

FileDownload / File: emd_3877.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAn 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.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.9 Å/pix.
x 180 pix.
= 342. Å		1.9 Å/pix.
x 180 pix.
= 342. Å		1.9 Å/pix.
x 180 pix.
= 342. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.9 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.09078771 - 0.12465287
Average (Standard dev.)0.00024227778 (±0.0091167325)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 342.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.91.91.9
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z342.000342.000342.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.0910.1250.000

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Supplemental data

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Sample components

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Entire : Bcs inner membrane sub-complex.

EntireName: Bcs inner membrane sub-complex.
Components
  • Complex: Bcs inner membrane sub-complex.

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Supramolecule #1: Bcs inner membrane sub-complex.

SupramoleculeName: 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.
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094 / Location in cell: inner membrane
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: Bl21(DE3) / Recombinant plasmid: pCDF-Duet
Molecular weightTheoretical: 1 MDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 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
StainingType: 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.
GridMaterial: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
DetailsSamples 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

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Number grids imaged: 2 / Average electron dose: 12.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.5 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 0.5 µm / Nominal magnification: 50000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 127400
Details: Particles were picked using the automated picking function of EMAN2.0/Boxer.
CTF correctionSoftware - Name: EMAN (ver. 2.0)
Details: CTF correction was carried out in EMAN2.0 (e2ctf) in two rounds, before and after structure factor generation
Startup modelType of model: OTHER
Details: Initial model was generated using EMAN2.0 (e2initialmodel) using a subset of high contrast class averages following 2D classification in Relion1.4.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 21.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 14362
Initial angle assignmentType: NOT APPLICABLE / Software - Name: EMAN (ver. 2.0)
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 200 / Software - Name: RELION (ver. 1.4)
Details: Particles were saved as an .mrcs stack and subjected to two rounds of 2D classification in Relion. Only high-contrast classes (20182 particles in total) were used for 3D classification with ...Details: Particles were saved as an .mrcs stack and subjected to two rounds of 2D classification in Relion. Only high-contrast classes (20182 particles in total) were used for 3D classification with three classes. Two of the classes converged to the same 3D model and the corresponding particles (14362) were used for 3D refinement of the EMAN2.0-generated initial model.
FSC plot (resolution estimation)

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