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- PDB-8c50: Pontibacter korlensis curli subunit CsgA -

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Basic information

Entry
Database: PDB / ID: 8c50
TitlePontibacter korlensis curli subunit CsgA
ComponentsCurlin associated repeat-containing protein
KeywordsPROTEIN FIBRIL / bacterial functional amyloid
Function / homologyCurlin associated / Curlin associated repeat / pilus / cell adhesion / Curlin associated repeat-containing protein
Function and homology information
Biological speciesPontibacter korlensis (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 7.6 Å
AuthorsRemaut, H. / Sleutel, M. / Pradhan, B.
Funding support Belgium, European Union, 3items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO)G0G0818N Belgium
Research Foundation - Flanders (FWO)G043021N Belgium
European Research Council (ERC)649082European Union
CitationJournal: Nat Commun / Year: 2023
Title: Structural analysis and architectural principles of the bacterial amyloid curli.
Authors: Mike Sleutel / Brajabandhu Pradhan / Alexander N Volkov / Han Remaut /
Abstract: Two decades have passed since the initial proposition that amyloids are not only (toxic) byproducts of an unintended aggregation cascade, but that they can also be produced by an organism to serve a ...Two decades have passed since the initial proposition that amyloids are not only (toxic) byproducts of an unintended aggregation cascade, but that they can also be produced by an organism to serve a defined biological function. That revolutionary idea was borne out of the realization that a large fraction of the extracellular matrix that holds Gram-negative cells into a persistent biofilm is composed of protein fibers (curli; tafi) with cross-β architecture, nucleation-dependent polymerization kinetics and classic amyloid tinctorial properties. The list of proteins shown to form so-called functional amyloid fibers in vivo has greatly expanded over the years, but detailed structural insights have not followed at a similar pace in part due to the associated experimental barriers. Here we combine extensive AlphaFold2 modelling and cryo-electron transmission microscopy to propose an atomic model of curli protofibrils, and their higher modes of organization. We uncover an unexpected structural diversity of curli building blocks and fibril architectures. Our results allow for a rationalization of the extreme physico-chemical robustness of curli, as well as earlier observations of inter-species curli promiscuity, and should facilitate further engineering efforts to expand the repertoire of curli-based functional materials.
History
DepositionJan 5, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 28, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Curlin associated repeat-containing protein
B: Curlin associated repeat-containing protein
C: Curlin associated repeat-containing protein


Theoretical massNumber of molelcules
Total (without water)114,1013
Polymers114,1013
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Curlin associated repeat-containing protein


Mass: 38033.785 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pontibacter korlensis (bacteria) / Gene: PKOR_12675 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: A0A0E3UX01

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: curli fiber of Pontibacteri korlensis CsgA / Type: COMPLEX
Details: Sample is generated by in vitro polymerisation of Pontibacter korlensis Csga subunits
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Pontibacter korlensis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21
Buffer solutionpH: 6 / Details: 15 mM MES pH 6.0
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal magnification: 60000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 800 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: JEOL CRYOSPECPORTER
Image recordingElectron dose: 64.66 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4455

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Processing

EM software
IDNameVersionCategory
1crYOLOparticle selection
2SerialEM3.0.8image acquisition
4CTFFIND4CTF correction
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 180 ° / Axial rise/subunit: 72 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 1817890
Details: 1000 filaments were manually boxed using e2helixboxer.py of the EMAN2 package and used as a training dataset for SPHIRE-crYOLO.
3D reconstructionResolution: 7.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 64138 / Symmetry type: HELICAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0017845
ELECTRON MICROSCOPYf_angle_d0.42110680
ELECTRON MICROSCOPYf_dihedral_angle_d10.0312736
ELECTRON MICROSCOPYf_chiral_restr0.0461161
ELECTRON MICROSCOPYf_plane_restr0.0011563

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