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- PDB-9i98: Cryo-EM structure of Shigella flexneri LptDE bound by a Bicyclic ... -

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Basic information

Entry
Database: PDB / ID: 9i98
TitleCryo-EM structure of Shigella flexneri LptDE bound by a Bicyclic peptide molecule (Compound 13)
Components
  • Compound 13 - Bicyclic Peptide binder
  • LPS-assembly lipoprotein LptE
  • LPS-assembly protein LptD
KeywordsMEMBRANE PROTEIN / outer membrane protein / lipid transport
Function / homology
Function and homology information


transporter complex / lipopolysaccharide transport / Gram-negative-bacterium-type cell outer membrane assembly / cell outer membrane / lipopolysaccharide binding
Similarity search - Function
LptD, C-terminal / LPS-assembly protein LptD / : / LPS transport system D / LPS-assembly lipoprotein LptE / Lipopolysaccharide-assembly / Organic solvent tolerance-like, N-terminal / LptA/(LptD N-terminal domain) LPS transport protein / Prokaryotic membrane lipoprotein lipid attachment site profile.
Similarity search - Domain/homology
: / LPS-assembly lipoprotein LptE / LPS-assembly protein LptD
Similarity search - Component
Biological speciesShigella flexneri (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsAllyjaun, S. / Newman, H. / Dunbar, E. / Hardwick, S.W. / Chirgadze, D.Y. / van den Berg, B. / Hubbard, J.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)U-016275 IAA-CiC United Kingdom
Innovate UK10086683 United Kingdom
CitationJournal: J Med Chem / Year: 2025
Title: High-Throughput Identification and Characterization of LptDE-Binding Bicycle Peptides Using Phage Display and Cryo-EM.
Authors: Shenaz Allyjaun / Emily Dunbar / Steven W Hardwick / Sarah Newell / Finn Holding / Catherine E Rowland / Megan A St Denis / Simone Pellegrino / Gustavo Arruda Bezerra / Nikolaos Bournakas / ...Authors: Shenaz Allyjaun / Emily Dunbar / Steven W Hardwick / Sarah Newell / Finn Holding / Catherine E Rowland / Megan A St Denis / Simone Pellegrino / Gustavo Arruda Bezerra / Nikolaos Bournakas / Dimitri Y Chirgadze / Lee Cooper / Giulia Paris / Nick Lewis / Peter Brown / Michael J Skynner / Michael J Dawson / Paul Beswick / Julia Hubbard / Bert van den Berg / Hector Newman /
Abstract: The lipopolysaccharide (LPS) transport (Lpt) system in Gram-negative bacteria maintains the integrity of the asymmetric bacterial outer membrane (OM). LPS biogenesis systems are essential in most ...The lipopolysaccharide (LPS) transport (Lpt) system in Gram-negative bacteria maintains the integrity of the asymmetric bacterial outer membrane (OM). LPS biogenesis systems are essential in most Gram-negative bacteria, with LptDE responsible for the delivery of LPS to the outer leaflet of the OM. As an externally accessible, essential protein, LptDE offers a promising target for inhibitor development without the need for cellular penetration. However, there are no direct inhibitors of LptDE, and drug discovery is made challenging since it is a membrane target without a conventional active site. Here, the bicycle phage display platform was used in combination with cryogenic-electron microscopy (cryo-EM) and surface plasmon resonance to identify and map bicyclic peptide binders to LptDE (SfLptDE). Four distinct epitopes with unique bicycle molecule binding motifs were identified across the SfLptD β-barrel. This method represents a streamlined workflow for the identification and prioritization of hit molecules against LptDE.
History
DepositionFeb 6, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 15, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: LPS-assembly protein LptD
B: LPS-assembly lipoprotein LptE
C: Compound 13 - Bicyclic Peptide binder
hetero molecules


Theoretical massNumber of molelcules
Total (without water)111,3147
Polymers109,4663
Non-polymers1,8484
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 LPS-assembly protein LptD


Mass: 87135.469 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Shigella flexneri (bacteria) / Gene: lptD, imp, ostA, SF0051, S0053 / Production host: Escherichia coli (E. coli) / References: UniProt: Q83SQ0
#2: Protein LPS-assembly lipoprotein LptE


Mass: 20338.121 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Shigella flexneri (bacteria) / Gene: lptE, rlpB, SF0640, S0662 / Production host: Escherichia coli (E. coli) / References: UniProt: Q83LX4
#3: Protein/peptide Compound 13 - Bicyclic Peptide binder


Mass: 1992.215 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Sugar ChemComp-LMT / DODECYL-BETA-D-MALTOSIDE


Type: D-saccharide / Mass: 510.615 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C24H46O11 / Comment: detergent*YM
#5: Chemical ChemComp-R06 / 1-[3,5-bis(2-chloranylethanoyl)-1,3,5-triazinan-1-yl]-2-chloranyl-ethanone


Mass: 316.569 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H12Cl3N3O3 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
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

ComponentName: S. flexneri LPS assembly complex LptDE bound to Bicyclic molecule Compound 13
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Shigella flexneri (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.8
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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 53.03 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 151742 / Symmetry type: POINT
RefinementHighest resolution: 2.86 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0046112
ELECTRON MICROSCOPYf_angle_d0.6688303
ELECTRON MICROSCOPYf_dihedral_angle_d6.313853
ELECTRON MICROSCOPYf_chiral_restr0.046879
ELECTRON MICROSCOPYf_plane_restr0.0081075

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