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- EMDB-10721: 2.85 A cryo-EM structure of the in vivo assembled type 1 pilus rod -

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Basic information

Entry
Database: EMDB / ID: EMD-10721
Title2.85 A cryo-EM structure of the in vivo assembled type 1 pilus rod
Map data
Sample
  • Complex: Type 1 pilus rod
    • Protein or peptide: Type-1 fimbrial protein, A chain
KeywordsFimA / pilus / monomer / subunit / pili / main structural subunit / high resolution / STRUCTURAL PROTEIN / cryo-EM / helical processing / RELION / Chaperone-usher pilus
Function / homologycell adhesion involved in single-species biofilm formation / Fimbrial-type adhesion domain / Fimbrial protein / Fimbrial-type adhesion domain superfamily / Adhesion domain superfamily / pilus / cell adhesion / identical protein binding / Type-1 fimbrial protein, A chain
Function and homology information
Biological speciesEscherichia coli (E. coli)
Methodhelical reconstruction / cryo EM / Resolution: 2.85 Å
AuthorsZyla D / Hospenthal M
Funding support Switzerland, 2 items
OrganizationGrant numberCountry
Swiss National Science Foundation310030B_176403/1 Switzerland
Swiss National Science Foundation31003A_156304 Switzerland
CitationJournal: Nat Commun / Year: 2024
Title: The assembly platform FimD is required to obtain the most stable quaternary structure of type 1 pili.
Authors: Dawid S Zyla / Thomas Wiegand / Paul Bachmann / Rafal Zdanowicz / Christoph Giese / Beat H Meier / Gabriel Waksman / Manuela K Hospenthal / Rudi Glockshuber /
Abstract: Type 1 pili are important virulence factors of uropathogenic Escherichia coli that mediate bacterial attachment to epithelial cells in the urinary tract. The pilus rod is comprised of thousands of ...Type 1 pili are important virulence factors of uropathogenic Escherichia coli that mediate bacterial attachment to epithelial cells in the urinary tract. The pilus rod is comprised of thousands of copies of the main structural subunit FimA and is assembled in vivo by the assembly platform FimD. Although type 1 pilus rods can self-assemble from FimA in vitro, this reaction is slower and produces structures with lower kinetic stability against denaturants compared to in vivo-assembled rods. Our study reveals that FimD-catalysed in vitro-assembled type 1 pilus rods attain a similar stability as pilus rods assembled in vivo. Employing structural, biophysical and biochemical analyses, we show that in vitro assembly reactions lacking FimD produce pilus rods with structural defects, reducing their stability against dissociation. Overall, our results indicate that FimD is not only required for the catalysis of pilus assembly, but also to control the assembly of the most stable quaternary structure.
History
DepositionMar 2, 2020-
Header (metadata) releaseMar 31, 2021-
Map releaseMar 31, 2021-
UpdateApr 17, 2024-
Current statusApr 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.021
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.021
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6y7s
  • Surface level: 0.021
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6y7s
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10721.png

Downloads & links

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Map

FileDownload / File: emd_10721.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.08187 Å
Density
Contour LevelBy AUTHOR: 0.021 / Movie #1: 0.021
Minimum - Maximum-0.025690159 - 0.059050027
Average (Standard dev.)-0.000023873441 (±0.0025369246)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.9587 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081871093751.081871093751.08187109375
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z276.959276.959276.959
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0260.059-0.000

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Supplemental data

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Mask #1

Fileemd_10721_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: #1

Fileemd_10721_additional_1.map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_10721_half_map_1.map
Projections & Slices
AxesZYX

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Histogram

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Half map: #1

Fileemd_10721_half_map_2.map
Projections & Slices
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Sample components

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Entire : Type 1 pilus rod

EntireName: Type 1 pilus rod
Components
  • Complex: Type 1 pilus rod
    • Protein or peptide: Type-1 fimbrial protein, A chain

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Supramolecule #1: Type 1 pilus rod

SupramoleculeName: Type 1 pilus rod / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Type 1 pili recombinantly expressed, assembled in vivo and subsequently purified from the E. coli cell surface.
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 20.3 kDa/nm

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Macromolecule #1: Type-1 fimbrial protein, A chain

MacromoleculeName: Type-1 fimbrial protein, A chain / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 18.121074 KDa
Recombinant expressionOrganism: Escherichia coli str. K-12 substr. W3110 (bacteria)
SequenceString:
MKIKTLAIVV LSALSLSSTA ALAAATTVNG GTVHFKGEVV NAACAVDAGS VDQTVQLGQV RTASLAQEGA TSSAVGFNIQ LNDCDTNVA SKAAVAFLGT AIDAGHTNVL ALQSSAAGSA TNVGVQILDR TGAALTLDGA TFSSETTLNN GTNTIPFQAR Y FATGAATP GAANADATFK VQYQ

UniProtKB: Type-1 fimbrial protein, A chain

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration1.58 mg/mL
BufferpH: 7 / Details: in ddH2O.
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 3 ul sample, 30 s wait time, 0.5 s drain time, 6 s blotting.

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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 defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / 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 grids imaged: 1 / Number real images: 3469 / Average exposure time: 8.0 sec. / Average electron dose: 50.4 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 516000
Details: Autopicking based on the 2D classes from manually chosen filaments
Startup modelType of model: OTHER / Details: featureless cylinder
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 3.08)
Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 7.85334 Å
Applied symmetry - Helical parameters - Δ&Phi: 115.001 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.08) / Details: local searches 0.9 degree with mask / Number images used: 40805
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5oh0


Chain - Chain ID: D / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 49.9714 / Target criteria: Correlation coefficient
Output model

PDB-6y7s:
2.85 A cryo-EM structure of the in vivo assembled type 1 pilus rod

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