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- EMDB-10206: Structure of the curli secretion-assembly complex CsgG:CsgF -

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Basic information

Entry
Database: EMDB / ID: EMD-10206
TitleStructure of the curli secretion-assembly complex CsgG:CsgF
Map data
Sample
  • Complex: CsgG:CsgF complex in DDM
    • Protein or peptide: Curli production assembly/transport component CsgF
    • Protein or peptide: Curli production assembly/transport component CsgG
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsVan der Verren SE / Remaut H
Funding support Belgium, 2 items
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, 2019-
Header (metadata) releaseJun 24, 2020-
Map releaseJun 24, 2020-
UpdateDec 16, 2020-
Current statusDec 16, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6si7
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10206.png

Downloads & links

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Map

FileDownload / File: emd_10206.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1 Å
Density
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.16426435 - 0.3008369
Average (Standard dev.)0.0007784759 (±0.008131751)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 280.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z280.000280.000280.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.1640.3010.001

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Supplemental data

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Sample components

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Entire : CsgG:CsgF complex in DDM

EntireName: CsgG:CsgF complex in DDM
Components
  • Complex: CsgG:CsgF complex in DDM
    • Protein or peptide: Curli production assembly/transport component CsgF
    • Protein or peptide: Curli production assembly/transport component CsgG

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Supramolecule #1: CsgG:CsgF complex in DDM

SupramoleculeName: CsgG:CsgF complex in DDM / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MC4100
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: C43 / Recombinant plasmid: pTRC99
Molecular weightTheoretical: 400 KDa

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Macromolecule #1: Curli production assembly/transport component CsgF

MacromoleculeName: Curli production assembly/transport component CsgF / type: protein_or_peptide / ID: 1 / Details: Only first 35 residues were visible and built / Number of copies: 9 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 13.744857 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GTMTFQFRNP NFGGNPNNGA FLLNSAQAQN SYKDPSYNDD FGIETPSALD NFTQAIQSQI LGGLLSNINT GKPGRMVTND YIVDIANRD GQLQLNVTDR KTGQTSTIQV SGLQNNSTDF HHHHHH

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Macromolecule #2: Curli production assembly/transport component CsgG

MacromoleculeName: Curli production assembly/transport component CsgG / type: protein_or_peptide / ID: 2 / Number of copies: 9 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 30.110193 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: CLTAPPKEAA RPTLMPRAQS YKDLTHLPAP TGKIFVSVYN IQDETGQFKP YPASNFSTAV PQSATAMLVT ALKDSRWFIP LERQGLQNL LNERKIIRAA QENGTVAINN RIPLQSLTAA NIMVEGSIIG YESNVKSGGV GARYFGIGAD TQYQLDQIAV N LRVVNVST ...String:
CLTAPPKEAA RPTLMPRAQS YKDLTHLPAP TGKIFVSVYN IQDETGQFKP YPASNFSTAV PQSATAMLVT ALKDSRWFIP LERQGLQNL LNERKIIRAA QENGTVAINN RIPLQSLTAA NIMVEGSIIG YESNVKSGGV GARYFGIGAD TQYQLDQIAV N LRVVNVST GEILSSVNTS KTILSYEVQA GVFRFIDYQR LLEGEVGYTS NEPVMLCLMS AIETGVIFLI NDGIDRGLWD LQ NKAERQN DILVKYRHMS VPPESSAWSH PQFEK

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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

Concentration0.03 mg/mL
BufferpH: 8
Component:
ConcentrationFormula
50.0 mMTris
200.0 mMNaClSodium chloride
0.03 %DDM
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 400 / Support film - Material: GRAPHENE OXIDE / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Instrument: GATAN CRYOPLUNGE 3

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 2045 / Average electron dose: 56.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1.8)
Details: Phase flipping is done internally during relion processing
Startup modelType of model: INSILICO MODEL
In silico model: Initial model was computed using e2initialmodel.py from the EMAN2 software
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final 3D classificationSoftware - Name: RELION (ver. 2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final reconstructionApplied symmetry - Point group: C9 (9 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 62000
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4uv3

Output model

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

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