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- PDB-8piz: Neisseria meningitidis Type IV pilus SB-DATDH variant bound to th... -

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Basic information

Entry
Database: PDB / ID: 8piz
TitleNeisseria meningitidis Type IV pilus SB-DATDH variant bound to the C24 nanobody
Components
  • C24 nanobody
  • Pilin
KeywordsPROTEIN FIBRIL / Pilin / Extracellular / Adhesion / Aggregation
Function / homology
Function and homology information


pilus / cell adhesion / membrane
Similarity search - Function
Fimbrial protein pilin / Pilin (bacterial filament) / : / Prokaryotic N-terminal methylation motif / Prokaryotic N-terminal methylation site / Pilin-like
Similarity search - Domain/homology
Chem-B6D / SN-GLYCEROL-3-PHOSPHATE / Pilin
Similarity search - Component
Biological speciesVicugna pacos (alpaca)
Neisseria meningitidis 8013 (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.75 Å
AuthorsFernandez-Martinez, D. / Dumenil, G.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR 18 CE11 0022 France
CitationJournal: Nat Commun / Year: 2024
Title: Cryo-EM structures of type IV pili complexed with nanobodies reveal immune escape mechanisms.
Authors: David Fernandez-Martinez / Youxin Kong / Sylvie Goussard / Agustin Zavala / Pauline Gastineau / Martial Rey / Gabriel Ayme / Julia Chamot-Rooke / Pierre Lafaye / Matthijn Vos / Ariel Mechaly ...Authors: David Fernandez-Martinez / Youxin Kong / Sylvie Goussard / Agustin Zavala / Pauline Gastineau / Martial Rey / Gabriel Ayme / Julia Chamot-Rooke / Pierre Lafaye / Matthijn Vos / Ariel Mechaly / Guillaume Duménil /
Abstract: Type IV pili (T4P) are prevalent, polymeric surface structures in pathogenic bacteria, making them ideal targets for effective vaccines. However, bacteria have evolved efficient strategies to evade ...Type IV pili (T4P) are prevalent, polymeric surface structures in pathogenic bacteria, making them ideal targets for effective vaccines. However, bacteria have evolved efficient strategies to evade type IV pili-directed antibody responses. Neisseria meningitidis are prototypical type IV pili-expressing Gram-negative bacteria responsible for life threatening sepsis and meningitis. This species has evolved several genetic strategies to modify the surface of its type IV pili, changing pilin subunit amino acid sequence, nature of glycosylation and phosphoforms, but how these modifications affect antibody binding at the structural level is still unknown. Here, to explore this question, we determine cryo-electron microscopy (cryo-EM) structures of pili of different sequence types with sufficiently high resolution to visualize posttranslational modifications. We then generate nanobodies directed against type IV pili which alter pilus function in vitro and in vivo. Cyro-EM in combination with molecular dynamics simulation of the nanobody-pilus complexes reveals how the different types of pili surface modifications alter nanobody binding. Our findings shed light on the impressive complementarity between the different strategies used by bacteria to avoid antibody binding. Importantly, we also show that structural information can be used to make informed modifications in nanobodies as countermeasures to these immune evasion mechanisms.
History
DepositionJun 22, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 3, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 9, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Pilin
B: C24 nanobody
hetero molecules


Theoretical massNumber of molelcules
Total (without water)33,7744
Polymers33,3552
Non-polymers4182
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: Protein Pilin


Mass: 17067.182 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Neisseria meningitidis 8013 (bacteria) / References: UniProt: A0A1I9GEU1
#2: Antibody C24 nanobody


Mass: 16288.223 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#3: Chemical ChemComp-G3P / SN-GLYCEROL-3-PHOSPHATE


Mass: 172.074 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H9O6P / Feature type: SUBJECT OF INVESTIGATION
#4: Sugar ChemComp-B6D / 2,4-bisacetamido-2,4,6-trideoxy-beta-D-glucopyranose / 2,4-bis(acetylamino)-2,4,6-trideoxy-beta-D-glucopyranose / 2,4-diacetamido-2,4,6-trideoxy-beta-D-glucopyranose / bacillosamine / 2,4-bisacetamido-2,4,6-trideoxy-beta-D-glucose / 2,4-bisacetamido-2,4,6-trideoxy-D-glucose / 2,4-bisacetamido-2,4,6-trideoxy-glucose


Type: D-saccharide, beta linking / Mass: 246.260 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H18N2O5 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
b-D-6-deoxy-GlcpNAc4NAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
Has ligand of interestY
Has protein modificationY

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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: Neisseria meningitidis PilE, SB-DATDH variant, bound to the C24 nanobody
Type: COMPLEX / Entity ID: #1-#2 / Source: NATURAL
Source (natural)Organism: Neisseria meningitidis 8013 (bacteria)
Buffer solutionpH: 7.4
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 100.7 ° / Axial rise/subunit: 10.449 Å / Axial symmetry: C1
3D reconstructionResolution: 2.75 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 249060 / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 51.34 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00182223
ELECTRON MICROSCOPYf_angle_d0.44443015
ELECTRON MICROSCOPYf_chiral_restr0.0384348
ELECTRON MICROSCOPYf_plane_restr0.0028383
ELECTRON MICROSCOPYf_dihedral_angle_d3.5694326

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