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- PDB-9d6f: Cryo-EM structure of E. coli FimH lectin domain bound to Fabs 440... -

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Basic information

Entry
Database: PDB / ID: 9d6f
TitleCryo-EM structure of E. coli FimH lectin domain bound to Fabs 440-2 and 454-3
Components
  • 440-2 Fab heavy chain
  • 440-2 Fab light chain
  • 445-3 Fab heavy chain
  • 445-3 Fab light chain
  • Type 1 fimbrin D-mannose specific adhesin FimH, Donor strand complemented with FimG peptide 'triple mutant'
KeywordsCELL ADHESION / SUGAR BINDING PROTEIN / Antigen / complex / adhesion / STRUCTURAL PROTEIN
Function / homology
Function and homology information


pilus tip / mechanosensory behavior / cell adhesion involved in single-species biofilm formation / pilus / cell-substrate adhesion / D-mannose binding / host cell membrane / cell adhesion
Similarity search - Function
FimH, mannose-binding domain / FimH, mannose binding / Fimbrial-type adhesion domain / Fimbrial protein / : / Fimbrial-type adhesion domain superfamily / Adhesion domain superfamily
Similarity search - Domain/homology
Protein FimG / Type 1 fimbrin D-mannose specific adhesin
Similarity search - Component
Biological speciesMus musculus (house mouse)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.24 Å
AuthorsLees, J.A. / Han, S.
Funding support United States, 1items
OrganizationGrant numberCountry
Other private United States
CitationJournal: PLoS Pathog / Year: 2025
Title: Structure-based design of an immunogenic, conformationally stabilized FimH antigen for a urinary tract infection vaccine.
Authors: Natalie C Silmon de Monerri / Ye Che / Joshua A Lees / Jayasankar Jasti / Huixian Wu / Matthew C Griffor / Srinivas Kodali / Julio Cesar Hawkins / Jacqueline Lypowy / Christopher Ponce / ...Authors: Natalie C Silmon de Monerri / Ye Che / Joshua A Lees / Jayasankar Jasti / Huixian Wu / Matthew C Griffor / Srinivas Kodali / Julio Cesar Hawkins / Jacqueline Lypowy / Christopher Ponce / Kieran Curley / Alexandre Esadze / Juan Carcamo / Thomas McLellan / David Keeney / Arthur Illenberger / Yury V Matsuka / Suman Shanker / Laurent Chorro / Alexey V Gribenko / Seungil Han / Annaliesa S Anderson / Robert G K Donald /
Abstract: Adhesion of E. coli to the urinary tract epithelium is a critical step in establishing urinary tract infections. FimH is an adhesin positioned on the fimbrial tip which binds to mannosylated proteins ...Adhesion of E. coli to the urinary tract epithelium is a critical step in establishing urinary tract infections. FimH is an adhesin positioned on the fimbrial tip which binds to mannosylated proteins on the urinary tract epithelium via its lectin domain (FimHLD). FimH is of interest as a target of vaccines to prevent urinary tract infections (UTI). Previously, difficulties in obtaining purified recombinant FimH from E. coli along with the poor inherent immunogenicity of FimH have hindered the development of effective FimH vaccine candidates. To overcome these challenges, we have devised a novel production method using mammalian cells to produce high yields of homogeneous FimH protein with comparable biochemical and immunogenic properties to FimH produced in E. coli. Next, to optimize conformational stability and immunogenicity of FimH, we used a computational approach to design improved FimH mutants and evaluated their biophysical and biochemical properties, and murine immunogenicity using a bacterial adhesion inhibition assay. This approach identified an immunogenic FimH variant (FimH-donor-strand complemented with FimG peptide 'triple mutant', FimH-DSG TM) capable of blocking bacterial adhesion that is produced at high yields in mammalian cells. By x-ray crystallography, we confirmed that the stabilized structure of the FimHLD in FimH-DSG TM is similar to native FimH on the fimbrial tip. Characterization of monoclonal antibodies elicited by FimH-DSG TM that can block bacterial binding to mannosylated surfaces identified 4 non-overlapping binding sites whose epitopes were mapped via a combinatorial cryogenic electron microscopy approach. Novel inhibitory epitopes in the lectin binding FimH were identified, revealing diverse functional mechanisms of FimH-directed antibodies with relevance to FimH-targeted UTI vaccines.
History
DepositionAug 15, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 5, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
L: 440-2 Fab light chain
H: 440-2 Fab heavy chain
A: 445-3 Fab heavy chain
C: 445-3 Fab light chain
D: Type 1 fimbrin D-mannose specific adhesin FimH, Donor strand complemented with FimG peptide 'triple mutant'


Theoretical massNumber of molelcules
Total (without water)128,9115
Polymers128,9115
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Antibody 440-2 Fab light chain


Mass: 24367.045 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Homo sapiens (human)
#2: Antibody 440-2 Fab heavy chain


Mass: 24603.318 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Homo sapiens (human)
#3: Antibody 445-3 Fab heavy chain


Mass: 24124.822 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): Homo sapiens / Production host: Homo sapiens (human)
#4: Antibody 445-3 Fab light chain


Mass: 23696.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Homo sapiens (human)
#5: Protein Type 1 fimbrin D-mannose specific adhesin FimH, Donor strand complemented with FimG peptide 'triple mutant'


Mass: 32118.596 Da / Num. of mol.: 1 / Mutation: N7S, G15A, G16A, V27A, N70S, N228Q within FimH
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: fimH, b4320, JW4283, fimG, b4319, JW4282 / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P08191, UniProt: P08190
Has protein modificationY

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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

Component
IDNameTypeEntity IDParent-IDSource
1FimH-DSG TM in complex with Fabs 440-2 and 445-3COMPLEXall0MULTIPLE SOURCES
2FimH-DSG TMCOMPLEX#51RECOMBINANT
3Fab 440-2COMPLEX#1-#21RECOMBINANT
4Fab 445-3COMPLEX#3-#41RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Mus musculus (house mouse)10090
44Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Cricetulus griseus (Chinese hamster)10029
33Homo sapiens (human)9606
44Homo sapiens (human)9606
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen 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 defocus max: 3200 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.24 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 185595 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0044786
ELECTRON MICROSCOPYf_angle_d0.8396509
ELECTRON MICROSCOPYf_dihedral_angle_d5.177657
ELECTRON MICROSCOPYf_chiral_restr0.049715
ELECTRON MICROSCOPYf_plane_restr0.007830

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