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- EMDB-3864: Negative stain electron microscopy reconstruction of a cellulose ... -

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Basic information

Entry
Database: EMDB / ID: EMD-3864
TitleNegative stain electron microscopy reconstruction of a cellulose secretion (Bcs) macrocomplex from E. coli
Map dataBcs macrocomplex encompassing most inner-membrane and cytosolic components of the E. coli cellulose secretion system. Negative-stain EM
Sample
  • Complex: Protein macrocomplex encompassing most cytosolic and inner-membrane components of the E. coli system for cellulose secretion
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / negative staining / Resolution: 16.7 Å
AuthorsKrasteva PV / Fronzes R
Funding support France, 4 items
OrganizationGrant numberCountry
CNRSATIP-Avenir 2016 France
FRMDEQ20140329508 France
French National Research AgencyANR-10-LABX-62-IBEID France
European Research CouncilMolStructTransfo 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 11, 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: 0.0104
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.0104
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3864.png

Downloads & links

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Map

FileDownload / File: emd_3864.map.gz / Format: CCP4 / Size: 35.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBcs macrocomplex encompassing most inner-membrane and cytosolic components of the E. coli cellulose secretion system. Negative-stain EM
Voxel sizeX=Y=Z: 1.9 Å
Density
Contour LevelBy AUTHOR: 0.0104 / Movie #1: 0.0104
Minimum - Maximum-0.07754264 - 0.13978234
Average (Standard dev.)0.00051137165 (±0.008628474)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions210210210
Spacing210210210
CellA=B=C: 399.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.91.91.9
M x/y/z210210210
origin x/y/z0.0000.0000.000
length x/y/z399.000399.000399.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS210210210
D min/max/mean-0.0780.1400.001

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Supplemental data

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Sample components

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Entire : Protein macrocomplex encompassing most cytosolic and inner-membra...

EntireName: Protein macrocomplex encompassing most cytosolic and inner-membrane components of the E. coli system for cellulose secretion
Components
  • Complex: Protein macrocomplex encompassing most cytosolic and inner-membrane components of the E. coli system for cellulose secretion

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Supramolecule #1: Protein macrocomplex encompassing most cytosolic and inner-membra...

SupramoleculeName: Protein macrocomplex encompassing most cytosolic and inner-membrane components of the E. coli system for cellulose secretion
type: complex / ID: 1 / Parent: 0
Details: Protein macrocomplex encompassing cytosolic (BcsRQEF) and inner-membrane (BcsAB) components. Isolated from purified and detergent-solubilised E. coli 1094 membranes using FLAG-tagged BcsA as ...Details: Protein macrocomplex encompassing cytosolic (BcsRQEF) and inner-membrane (BcsAB) components. Isolated from purified and detergent-solubilised E. coli 1094 membranes using FLAG-tagged BcsA as bait. Stabilised by gentle glutaraldehyde cross-linking over a density gradient (GraFix).
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: 1094 2K7 BcsA-HA-FLAG / Recombinant plasmid: Chromosome-driven expression
Molecular weightTheoretical: 1.4 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 pH 8.0 120 mM NaCl, 10%-40% glycerol, 5 mM MgCl2 10 uM AppCp 2 uM cyclic c-di-GMP cOmplete protease inhibitors
StainingType: NEGATIVE / Material: Uranyl Acetate
Details: 5 micrometers of sample were spotted on glow-discharged carbon-coated copper grids. After 1 minutes the liquid was blotted off and the sample was stained by quick passage through 3 drops of ...Details: 5 micrometers of sample were spotted on glow-discharged carbon-coated copper grids. After 1 minutes the liquid was blotted off and the sample was stained by quick passage through 3 drops of 2% uranyl acetate. The sample was left for 20-30 seconds on the last drop of stain, after which the liquid was blotted off and the sample was allowed to air-dry
GridMaterial: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: 7mA, 10 microns
DetailsSample was purified from pelleted and solubilised membrane fractions of the E. coli 1094 2K7 BcsA-HA-FLAG strain using anti-FLAG affinity gel, 3xFLAG peptide elution and glycerol gradient purification coupled with gentle glutaraldehyde cross-linking (GraFix). Sample was monodisperse and subjected to thin-layer uranyl acetate staining.

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus min: 0.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Number grids imaged: 2 / Average electron dose: 12.0 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 44056
CTF correctionSoftware - Name: EMAN (ver. 2.0)
Details: CTF correction was performed in EMAN2.0 (e2ctf) using the automated fitting function before and after structure factor generation
Startup modelType of model: OTHER
Details: An initial 3D model was generated in EMAN2.0 using the e2initialmodel function.
Initial angle assignmentType: OTHER / Software - Name: EMAN (ver. 2.0)
Details: An initial 3D model was generated in EMAN2.0 using the e2initialmodel function. Additional initial models were generated by random conical tilt reconstruction in EMAN using a separate ...Details: An initial 3D model was generated in EMAN2.0 using the e2initialmodel function. Additional initial models were generated by random conical tilt reconstruction in EMAN using a separate dataset collected in tilt-pairs.
Final 3D classificationNumber classes: 200 / Software - Name: RELION (ver. 1.4)
Details: A total of 44056 particles were subjected to two rounds of 2D classification in RELION using 200 classes for each. After each round, low-contrast or smeared classes were rejected. A total of ...Details: A total of 44056 particles were subjected to two rounds of 2D classification in RELION using 200 classes for each. After each round, low-contrast or smeared classes were rejected. A total of 24769 particles were saved for 3D classification in RELION using 3 classes. As all 3D classes showed similar features the EMAN2.0-generated initial model was refined against all 24769 particles.
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 16.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 24769
FSC plot (resolution estimation)

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