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- PDB-9rqi: Cryo-EM structure of Shigella flexneri LptDE bound by phage RBP r... -

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Basic information

Entry
Database: PDB / ID: 9rqi
TitleCryo-EM structure of Shigella flexneri LptDE bound by phage RBP reveals N-terminal strand insertion into lateral gate
Components
  • LPS-assembly lipoprotein LptE
  • LPS-assembly protein LptD
  • Tail fiber protein
KeywordsMEMBRANE PROTEIN / lipopolysaccharide transport / receptor-binding protein / outer membrane complex / beta-barrel / bacteriophage
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
Protein of unknown function DUF6453 / Family of unknown function (DUF6453) / 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
Tail fiber protein / LPS-assembly lipoprotein LptE / LPS-assembly protein LptD
Similarity search - Component
Biological speciesShigella flexneri (bacteria)
Escherichia phage Oekolampad (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.76 Å
AuthorsDunbar, E. / Basle, A. / van den Berg, B.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)U-016275 IAA-CiC United Kingdom
Other private
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Small siphophage binding to an open state of the LptDE outer membrane lipopolysaccharide translocon.
Authors: Emily Dunbar / Robert Clark / Arnaud Baslé / Shenaz Allyjaun / Hector Newman / Julia Hubbard / Syma Khalid / Bert van den Berg /
Abstract: Bacteriophages are bacterial viruses that provide alternatives to small-molecule drugs to combat infections by antibiotic-resistant bacteria. To infect a bacterial host, a phage needs to bind to the ...Bacteriophages are bacterial viruses that provide alternatives to small-molecule drugs to combat infections by antibiotic-resistant bacteria. To infect a bacterial host, a phage needs to bind to the bacterial surface via receptor binding proteins (RBPs), which are critical for determining host specificity. For functionally important receptors, the RBP-receptor interaction could be exploited via phage steering, where emerging bacterial resistance due to receptor modification could make bacteria less fit or virulent. Despite this, relatively little is known about RBP-receptor interactions. Here, we build on the recent discovery of coliphages that have the outer membrane (OM) lipopolysaccharide translocon LptDE as their terminal receptor and show via cryogenic electron microscopy that, surprisingly, the RBP of the small siphophage Oekolampad binds to a hitherto unobserved, open state of LptDE. The open lateral gate of LptD is occupied by a β-strand peptide originating from the degraded N-terminal jellyroll domain of LptD, suggesting the possibility of LptD inhibition via peptidomimetics. A structure of LptDE in complex with the superinfection exclusion (SE) protein Rtp45 of the Oekolampad-related phage Rtp shows a mechanism of SE where Rtp45-induced conformational changes in LptD resulting from steric clashes preclude RBP binding. Finally, analysis of spontaneous Oekolampad-resistant mutants identifies mutations in LptD that abolish the LptDE-RBP interaction in vitro. SDS-EDTA sensitivity assays of the mutants show no major OM defects, consistent with largely preserved LptDE function, and suggesting that phage steering via LptDE might be challenging.
History
DepositionJun 25, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 10, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: LPS-assembly protein LptD
B: LPS-assembly lipoprotein LptE
C: Tail fiber protein


Theoretical massNumber of molelcules
Total (without water)147,2953
Polymers147,2953
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein LPS-assembly protein LptD


Mass: 89740.602 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: 22206.471 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 Tail fiber protein


Mass: 35347.891 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage Oekolampad (virus) / Gene: bas18_0025 / Production host: Escherichia coli (E. coli) / References: UniProt: A0AAE8B389
Has protein modificationN

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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
1LptDE-RBP complexCOMPLEXall0RECOMBINANT
2LptDECOMPLEX#1-#21RECOMBINANT
3RBPCOMPLEX#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.1418 MDaNO
210.1073 MDaNO
310.0345 MDaNO
410.087 MDaNO
510.0203 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Shigella flexneri (bacteria)623
32Shigella flexneri (bacteria)623
43Escherichia phage Oekolampad (virus)2851982
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32Escherichia coli (E. coli)562
43Escherichia coli (E. coli)562
Buffer solutionpH: 7.5 / Details: 10mM HEPES, pH7.5, 100mM NaCl, 0.05% (w/v) DDM
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPESC8H18N2O4S1
2100 mMsodium chlorideNaCl1
30.05 (w/v) %DDMC24H46O111
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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 magnification: 165000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Image recordingAverage exposure time: 3.46 sec. / Electron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7000

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
4cryoSPARCCTF correction
7PHENIXmodel fitting
13Cootmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1508214
3D reconstructionResolution: 2.76 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 158797 / Symmetry type: POINT
Atomic model buildingSource name: AlphaFold / Type: in silico model

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