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- PDB-6si7: Structure of the curli secretion-assembly complex CsgG:CsgF -

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Basic information

Entry
Database: PDB / ID: 6si7
TitleStructure of the curli secretion-assembly complex CsgG:CsgF
Components
  • Curli production assembly/transport component CsgF
  • Curli production assembly/transport component CsgG
KeywordsPROTEIN TRANSPORT / Secretion Channel / Curli / Outer Membrane Protein / Nanopore Sensing / Bacterial amyloid
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
Type VIII secretion system, CsgF / Type VIII secretion system (T8SS), CsgF protein / 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 CsgF / Curli production assembly/transport component CsgG
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsVan der Verren, S.E. / Remaut, H.
Funding support Belgium, 2items
OrganizationGrant numberCountry
European Research Council649082 Belgium
Research Foundation - Flanders Belgium
CitationJournal: Nat Biotechnol / Year: 2020
Title: A dual-constriction biological nanopore resolves homonucleotide sequences with high fidelity.
Authors: Sander E Van der Verren / Nani Van Gerven / Wim Jonckheere / Richard Hambley / Pratik Singh / John Kilgour / Michael Jordan / E Jayne Wallace / Lakmal Jayasinghe / Han Remaut /
Abstract: Single-molecule long-read DNA sequencing with biological nanopores is fast and high-throughput but suffers reduced accuracy in homonucleotide stretches. We now combine the CsgG nanopore with the 35- ...Single-molecule long-read DNA sequencing with biological nanopores is fast and high-throughput but suffers reduced accuracy in homonucleotide stretches. We now combine the CsgG nanopore with the 35-residue N-terminal region of its extracellular interaction partner CsgF to produce a dual-constriction pore with improved signal and base-calling accuracy for homopolymer regions. The electron cryo-microscopy structure of CsgG in complex with full-length CsgF shows that the 33 N-terminal residues of CsgF bind inside the β-barrel of the pore, forming a defined second constriction. In complexes of CsgG bound to a 35-residue CsgF constriction peptide, the second constriction is separated from the primary constriction by ~25 Å. We find that both constrictions contribute to electrical signal modulation during single-stranded DNA translocation. DNA sequencing using a prototype CsgG-CsgF protein pore with two constrictions improved single-read accuracy by 25 to 70% in homopolymers up to 9 nucleotides long.
History
DepositionAug 8, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 24, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 15, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Nov 18, 2020Group: Structure summary / Category: struct_keywords / Item: _struct_keywords.text
Revision 1.3Dec 16, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4May 22, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-10206
  • Imaged by UCSF Chimera
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Assembly

Deposited unit
A: Curli production assembly/transport component CsgF
P: Curli production assembly/transport component CsgG
B: Curli production assembly/transport component CsgF
J: Curli production assembly/transport component CsgG
C: Curli production assembly/transport component CsgF
K: Curli production assembly/transport component CsgG
D: Curli production assembly/transport component CsgF
L: Curli production assembly/transport component CsgG
E: Curli production assembly/transport component CsgF
M: Curli production assembly/transport component CsgG
F: Curli production assembly/transport component CsgF
N: Curli production assembly/transport component CsgG
G: Curli production assembly/transport component CsgF
O: Curli production assembly/transport component CsgG
H: Curli production assembly/transport component CsgF
Q: Curli production assembly/transport component CsgG
I: Curli production assembly/transport component CsgF
R: Curli production assembly/transport component CsgG


Theoretical massNumber of molelcules
Total (without water)394,69518
Polymers394,69518
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: gel filtration, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area87730 Å2
ΔGint-433 kcal/mol
Surface area74500 Å2
MethodPISA

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Components

#1: Protein
Curli production assembly/transport component CsgF


Mass: 13744.857 Da / Num. of mol.: 9
Source method: isolated from a genetically manipulated source
Details: Only first 35 residues were visible and built / Source: (gene. exp.) Escherichia coli (E. coli) / Gene: csgF, b1038, JW1021 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43 / References: UniProt: P0AE98
#2: Protein
Curli production assembly/transport component CsgG


Mass: 30110.193 Da / Num. of mol.: 9
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: csgG, b1037, JW1020 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43 / References: UniProt: P0AEA2

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: CsgG:CsgF complex in DDM / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.40 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MC4100
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: C43 / Plasmid: pTRC99
Buffer solutionpH: 8
Buffer component
IDConc.FormulaBuffer-ID
150 mMTris1
2200 mMNaCl1
30.03 %DDM1
SpecimenConc.: 0.03 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 56 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 2045

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Processing

EM software
IDNameVersionCategory
1Gautomatchparticle selection
4CTFFIND4.1.8CTF correction
7UCSF Chimera1.10.2model fitting
8Coot0.8.1model fitting
10RELION2initial Euler assignment
11RELION2final Euler assignment
12RELION2classification
13RELION23D reconstruction
20PHENIX1.14model refinement
CTF correctionDetails: Phase flipping is done internally during relion processing
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C9 (9 fold cyclic)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 62000 / Symmetry type: POINT
Atomic model buildingPDB-ID: 4UV3

4uv3


Accession code: 4UV3 / Source name: PDB / Type: experimental model

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