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- PDB-9q8n: Cryo-EM structure of Shigella flexneri LptDE bound by a Bicyclic ... -

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Basic information

Entry
Database: PDB / ID: 9q8n
TitleCryo-EM structure of Shigella flexneri LptDE bound by a Bicyclic peptide molecule (Compound 16)
Components
  • (LPS-assembly ...) x 2
  • Compound 16 - Bicyclic Peptide Binder
KeywordsMEMBRANE PROTEIN / Lipid transport / Outer membrane protein
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
: / PALMITIC ACID / (2S)-3-hydroxypropane-1,2-diyl dihexadecanoate / 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: 2.54 Å
AuthorsAllyjaun, S. / Newman, H. / Chirgadze, D.Y. / Hardwick, S.W. / Hubbard, J. / van den Berg, B. / Dunbar, E.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Innovate UK10086683 United Kingdom
Medical Research Council (MRC, United Kingdom)U-016275 IAA-CiC 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 25, 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 16 - Bicyclic Peptide Binder
hetero molecules


Theoretical massNumber of molelcules
Total (without water)108,2998
Polymers105,9853
Non-polymers2,3135
Water00
1


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

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Components

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LPS-assembly ... , 2 types, 2 molecules AB

#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 BL21 (bacteria) / References: UniProt: Q83SQ0
#2: Protein LPS-assembly lipoprotein LptE


Mass: 17002.602 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 BL21 (bacteria) / References: UniProt: Q83LX4

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Protein/peptide / Sugars , 2 types, 3 molecules C

#3: Protein/peptide Compound 16 - Bicyclic Peptide Binder


Mass: 1847.186 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.: 2 / Source method: obtained synthetically / Formula: C24H46O11 / Comment: detergent*YM

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Non-polymers , 3 types, 3 molecules

#5: Chemical ChemComp-PLM / PALMITIC ACID


Mass: 256.424 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H32O2 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-Z41 / (2S)-3-hydroxypropane-1,2-diyl dihexadecanoate


Mass: 568.911 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C35H68O5 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-A1I4O / 1,3,5-tris(2-chloroethylsulfonyl)-1,3,5-triazinane


Mass: 466.810 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H18Cl3N3O6S3 / Feature type: SUBJECT OF INVESTIGATION

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Details

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: Cryo-EM structure of S. flexneri LptDE complex bound by a Bicyclic peptide molecule (Compound 8)
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.19 MDa / Experimental value: YES
Source (natural)Organism: Shigella flexneri (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 8 mg/ml / Embedding 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: 1800 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 12.06 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1Warpparticle selection
2PHENIX1.20.1_4487model refinement
10cryoSPARCv4.6.0initial Euler assignment
11cryoSPARCv4.6.0final Euler assignment
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 168619 / Symmetry type: POINT
RefinementHighest resolution: 2.54 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0056210
ELECTRON MICROSCOPYf_angle_d0.7138431
ELECTRON MICROSCOPYf_dihedral_angle_d7.215889
ELECTRON MICROSCOPYf_chiral_restr0.048885
ELECTRON MICROSCOPYf_plane_restr0.0071089

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