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- EMDB-17683: Neisseria meningitidis Type IV pilus SB-GATDH variant bound to th... -

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Basic information

Entry
Database: EMDB / ID: EMD-17683
TitleNeisseria meningitidis Type IV pilus SB-GATDH variant bound to the C24 nanobody
Map dataRaw map of the SB-GATDH variant of Neisseria meningitidis T4P bound to the C24 nanobody
Sample
  • Complex: Complex of Neisseria meningitidis PilE SB-GATDH and the C24 nanobody
    • Protein or peptide: Pilin
    • Protein or peptide: C24 nanobody
  • Ligand: SN-GLYCEROL-3-PHOSPHATE
  • Ligand: (2~{R})-~{N}-[(2~{R},3~{S},4~{S},5~{R},6~{R})-5-acetamido-2-methyl-4,6-bis(oxidanyl)oxan-3-yl]-2,3-bis(oxidanyl)propanamide
KeywordsPilin / Extracellular / Adhesion / Aggregation / PROTEIN FIBRIL
Function / homologyFimbrial protein pilin / Pilin (bacterial filament) / Prokaryotic N-terminal methylation motif / Prokaryotic N-terminal methylation site / Pilin-like / pilus / cell adhesion / membrane / Pilin
Function and homology information
Biological speciesNeisseria meningitidis 8013 (bacteria) / Vicugna pacos (alpaca)
Methodhelical reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsFernandez-Martinez D / Dumenil G
Funding support France, 1 items
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 21, 2023-
Header (metadata) releaseApr 3, 2024-
Map releaseApr 3, 2024-
UpdateApr 3, 2024-
Current statusApr 3, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17683.png

Downloads & links

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Map

FileDownload / File: emd_17683.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRaw map of the SB-GATDH variant of Neisseria meningitidis T4P bound to the C24 nanobody
Voxel sizeX=Y=Z: 0.93 Å
Density
Contour LevelBy AUTHOR: 0.103
Minimum - Maximum-0.14127703 - 0.5166042
Average (Standard dev.)0.00094258925 (±0.022493621)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 372.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17683_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: DL sharpened map of the SB-GATDH variant of...

Fileemd_17683_additional_1.map
AnnotationDL sharpened map of the SB-GATDH variant of Neisseria meningitidis T4P bound to the C24 nanobody
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A of the SB-GATDH variant of...

Fileemd_17683_half_map_1.map
AnnotationHalf map A of the SB-GATDH variant of Neisseria meningitidis T4P bound to the C24 nanobody
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B of the SB-GATDH variant of...

Fileemd_17683_half_map_2.map
AnnotationHalf map B of the SB-GATDH variant of Neisseria meningitidis T4P bound to the C24 nanobody
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of Neisseria meningitidis PilE SB-GATDH and the C24 nanobody

EntireName: Complex of Neisseria meningitidis PilE SB-GATDH and the C24 nanobody
Components
  • Complex: Complex of Neisseria meningitidis PilE SB-GATDH and the C24 nanobody
    • Protein or peptide: Pilin
    • Protein or peptide: C24 nanobody
  • Ligand: SN-GLYCEROL-3-PHOSPHATE
  • Ligand: (2~{R})-~{N}-[(2~{R},3~{S},4~{S},5~{R},6~{R})-5-acetamido-2-methyl-4,6-bis(oxidanyl)oxan-3-yl]-2,3-bis(oxidanyl)propanamide

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Supramolecule #1: Complex of Neisseria meningitidis PilE SB-GATDH and the C24 nanobody

SupramoleculeName: Complex of Neisseria meningitidis PilE SB-GATDH and the C24 nanobody
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Neisseria meningitidis 8013 (bacteria)

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Macromolecule #1: Pilin

MacromoleculeName: Pilin / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Neisseria meningitidis 8013 (bacteria)
Molecular weightTheoretical: 17.067182 KDa
Recombinant expressionOrganism: Neisseria meningitidis 8013 (bacteria)
SequenceString:
FTLIELMIVI AIVGILAAVA LPAYQDYTAR AQVSEAILLA EGQKSAVTEY YLNHGEWPGD NSSAGVATSA DIKGKYVQSV TVANGVITA QMASSNVNNE IKSKKLSLWA KRQNGSVKWF CGQPVTRTTA TATDVAAANG KTDDKINTKH LPSTCRDDSS A S

UniProtKB: Pilin

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Macromolecule #2: C24 nanobody

MacromoleculeName: C24 nanobody / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Molecular weightTheoretical: 13.911585 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString:
MAQLQLVESG GGLVQPGGSL RLSCASSGFR SDYYAIVWFR QAPGKEREGV SCISTSGKTT IYADSVKGRF TISRDNAKNT VYLQMNSLK PEDTAVYYCA ADFRGSRLSD VCSYSSMDYW GKGTLATVS

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Macromolecule #3: SN-GLYCEROL-3-PHOSPHATE

MacromoleculeName: SN-GLYCEROL-3-PHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: G3P
Molecular weightTheoretical: 172.074 Da
Chemical component information

ChemComp-G3P:
SN-GLYCEROL-3-PHOSPHATE / Glycerol 3-phosphate

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Macromolecule #4: (2~{R})-~{N}-[(2~{R},3~{S},4~{S},5~{R},6~{R})-5-acetamido-2-methy...

MacromoleculeName: (2~{R})-~{N}-[(2~{R},3~{S},4~{S},5~{R},6~{R})-5-acetamido-2-methyl-4,6-bis(oxidanyl)oxan-3-yl]-2,3-bis(oxidanyl)propanamide
type: ligand / ID: 4 / Number of copies: 1 / Formula: WKE
Molecular weightTheoretical: 292.286 Da

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.6 µm
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE / Details: Ab-initio
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Helical parameters - Δz: 10.429 Å
Applied symmetry - Helical parameters - Δ&Phi: 100.753 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 434690
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementProtocol: FLEXIBLE FIT
Output model

PDB-8pij:
Neisseria meningitidis Type IV pilus SB-GATDH variant bound to the C24 nanobody

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