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- EMDB-2750: Structure of the CsgG-CsgE complex -

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Basic information

Entry
Database: EMDB / ID: EMD-2750
TitleStructure of the CsgG-CsgE complex
Map datareconstruction of the CsgG-CsgE complex
Sample
  • Sample: CsgG-CsgE complex
  • Protein or peptide: CsgG
  • Protein or peptide: CsgE
KeywordsBacterial secretion / membrane protein
Function / homology
Function and homology information


: / curli secretion complex / curli assembly / protein secretion by the type VIII secretion system / protein transmembrane transport / single-species biofilm formation / cell outer membrane / outer membrane-bounded periplasmic space / identical protein binding / plasma membrane
Similarity search - Function
Curli assembly protein CsgE / Curli assembly protein CsgE / Curli assembly protein CsgE / Curli production assembly/transport component CsgG / Curli production assembly/transport component CsgG / Curli production assembly/transport component CsgG / Prokaryotic membrane lipoprotein lipid attachment site profile.
Similarity search - Domain/homology
Curli production assembly/transport component CsgE / Curli production assembly/transport component CsgG
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 24.0 Å
AuthorsKrasteva PV / Gubellini F / Remaut H / Fronzes R
CitationJournal: Nature / Year: 2014
Title: Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG.
Authors: Parveen Goyal / Petya V Krasteva / Nani Van Gerven / Francesca Gubellini / Imke Van den Broeck / Anastassia Troupiotis-Tsaïlaki / Wim Jonckheere / Gérard Péhau-Arnaudet / Jerome S Pinkner ...Authors: Parveen Goyal / Petya V Krasteva / Nani Van Gerven / Francesca Gubellini / Imke Van den Broeck / Anastassia Troupiotis-Tsaïlaki / Wim Jonckheere / Gérard Péhau-Arnaudet / Jerome S Pinkner / Matthew R Chapman / Scott J Hultgren / Stefan Howorka / Rémi Fronzes / Han Remaut /
Abstract: Curli are functional amyloid fibres that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria (predominantly of the α ...Curli are functional amyloid fibres that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria (predominantly of the α and γ classes). They provide a fitness advantage in pathogenic strains and induce a strong pro-inflammatory response during bacteraemia. Curli formation requires a dedicated protein secretion machinery comprising the outer membrane lipoprotein CsgG and two soluble accessory proteins, CsgE and CsgF. Here we report the X-ray structure of Escherichia coli CsgG in a non-lipidated, soluble form as well as in its native membrane-extracted conformation. CsgG forms an oligomeric transport complex composed of nine anticodon-binding-domain-like units that give rise to a 36-stranded β-barrel that traverses the bilayer and is connected to a cage-like vestibule in the periplasm. The transmembrane and periplasmic domains are separated by a 0.9-nm channel constriction composed of three stacked concentric phenylalanine, asparagine and tyrosine rings that may guide the extended polypeptide substrate through the secretion pore. The specificity factor CsgE forms a nonameric adaptor that binds and closes off the periplasmic face of the secretion channel, creating a 24,000 Å(3) pre-constriction chamber. Our structural, functional and electrophysiological analyses imply that CsgG is an ungated, non-selective protein secretion channel that is expected to employ a diffusion-based, entropy-driven transport mechanism.
History
DepositionAug 5, 2014-
Header (metadata) releaseAug 13, 2014-
Map releaseSep 24, 2014-
UpdateDec 10, 2014-
Current statusDec 10, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2750.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationreconstruction of the CsgG-CsgE complex
Voxel sizeX=Y=Z: 1.99 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.02745587 - 0.32763493
Average (Standard dev.)0.00198865 (±0.0180604)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-80-80-80
Dimensions160160160
Spacing160160160
CellA=B=C: 318.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.991.991.99
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z318.400318.400318.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ442626
MAP C/R/S123
start NC/NR/NS-80-80-80
NC/NR/NS160160160
D min/max/mean-0.0270.3280.002

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Supplemental data

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Sample components

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Entire : CsgG-CsgE complex

EntireName: CsgG-CsgE complex
Components
  • Sample: CsgG-CsgE complex
  • Protein or peptide: CsgG
  • Protein or peptide: CsgE

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Supramolecule #1000: CsgG-CsgE complex

SupramoleculeName: CsgG-CsgE complex / type: sample / ID: 1000
Details: The sample was reconstituted from purified components in vitro.
Oligomeric state: One nonamer of CsgG bound to one nonamer of CsgE
Number unique components: 2
Molecular weightTheoretical: 400 KDa

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Macromolecule #1: CsgG

MacromoleculeName: CsgG / type: protein_or_peptide / ID: 1
Name.synonym: Curli production assembly/transport component CsgG
Details: Strep-tagged / Number of copies: 9 / Oligomeric state: nonamer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: MC4100 / Location in cell: Outer membrane
Molecular weightTheoretical: 290 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pPG1 (pASK-IBA3plus derivative)
SequenceUniProtKB: Curli production assembly/transport component CsgG
GO: GO: 0022610, single-species biofilm formation, protein transmembrane transport, cell outer membrane
InterPro: Curli production assembly/transport component CsgG

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Macromolecule #2: CsgE

MacromoleculeName: CsgE / type: protein_or_peptide / ID: 2
Name.synonym: Curli production assembly/transport component CsgE
Details: Poly-histidine tagged. Used for CsgE-CsgG complex reconstitution and pull-down on Talon metal affinity resin.
Number of copies: 9 / Oligomeric state: nonamer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: MC4100 / Location in cell: Periplasm
Molecular weightTheoretical: 110 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: NEBC2566 / Recombinant plasmid: pNH27 (pET11d derivative)
SequenceUniProtKB: Curli production assembly/transport component CsgE
GO: GO: 0022610, single-species biofilm formation, protein transmembrane transport, cell outer membrane
InterPro: Curli assembly protein CsgE

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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

Concentration0.05 mg/mL
BufferpH: 8
Details: 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 100 mM imidazole, 2.5% xylitol, 0.2 mM DTT
GridDetails: Quantifoil R2/2 carbon coated grids (Quantifoil Micro Tools GmbH)
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 92 K / Instrument: LEICA EM GP / Method: Blot for 0.5 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 70350 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 95 K
Alignment procedureLegacy - Astigmatism: Astigmatism was checked during imaging and during CTF correction of the micrographs. Micrographs is astigmatism >10% were discarded.
DateJun 2, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 75 / Average electron dose: 20 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each micrograph
Final two d classificationNumber classes: 125
Final reconstructionApplied symmetry - Point group: C9 (9 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: OTHER / Software - Name: IMAGIC / Number images used: 1221
DetailsParticles were automatically selected from CTF-corrected micrographs using BOXER (EMAN2).

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