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- PDB-9k4b: Cryo-EM structure of depolymerase S2-4 from Klebsiella phage K64-1 -

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Basic information

Entry
Database: PDB / ID: 9k4b
TitleCryo-EM structure of depolymerase S2-4 from Klebsiella phage K64-1
ComponentsDepolymerase, capsule K1-specific
KeywordsANTIMICROBIAL PROTEIN / Depolymerase / Degradation of host capsule
Function / homology
Function and homology information


symbiont entry into host cell via disruption of host cell glycocalyx / symbiont entry into host cell via disruption of host cell envelope / virus tail / adhesion receptor-mediated virion attachment to host cell / lyase activity
Similarity search - Function
: / : / K1 capsule-specific polysaccharide lyase, C-terminal domain / K1 capsule-specific polysaccharide lyase, Rider domain / Right handed beta helix domain / Right handed beta helix region / Parallel beta-helix repeat / Parallel beta-helix repeats / Pectin lyase fold / Pectin lyase fold/virulence factor
Similarity search - Domain/homology
Depolymerase, capsule K1-specific
Similarity search - Component
Biological speciesKlebsiella phage K64-1 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.18 Å
AuthorsZhao, R. / Du, T. / Ren, Z. / Gu, J. / Ru, H.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32072824 China
National Natural Science Foundation of China (NSFC)32222083 China
CitationJournal: Microbiol Res / Year: 2026
Title: Characterization of the phage ΦK64 depolymerase S2-4 and its therapeutic effect against K1 serotype Klebsiella pneumoniae.
Authors: Rihong Zhao / Tianjiao Du / Yalu Ji / Zhuolu Ren / Shanshan Jiang / Heng Ru / Jingmin Gu /
Abstract: The infection rate and drug resistance of Klebsiella pneumoniae containing capsular polysaccharides (CPSs) have been increasing annually, resulting in severe human and animal infections. ...The infection rate and drug resistance of Klebsiella pneumoniae containing capsular polysaccharides (CPSs) have been increasing annually, resulting in severe human and animal infections. Depolymerases derived from bacteriophages can degrade CPSs and thus hold potential for treating bacterial infections. However, little is known about the mechanism by which K. pneumoniae phage depolymerases hydrolyze CPSs. In this study, the S2-4 encoded by the phage ΦK64 was identified as a potent depolymerase against K1 serotype Klebsiella CPSs. Cryo-electron microscopy structural analysis revealed that S2-4 forms a homotrimer with a spindle-like structure comprising a particle-binding domain, a core receptor-binding domain, an insertion domain, and a C-terminal domain. The results of structural assays suggest that S2-4 possesses multiple catalytic centers, which may contribute to its potent depolymerase activity. S2-4 inhibited K. pneumoniae biofilm formation, disrupted preformed biofilms, and enhanced macrophage adhesion and phagocytosis in depolymerase-treated bacteria. In a murine model, a single dose of 5 µg of S2-4 provided full protection against bacterial infection, underscoring the potent depolymerase activity of S2-4. These results indicate that S2-4 is a potent depolymerase against K1 serotype Klebsiella CPSs and has the potential to be a promising candidate for the clinical control of K. pneumoniae infections.
History
DepositionOct 21, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 25, 2026Provider: repository / Type: Initial release
Revision 1.0Mar 25, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 25, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 25, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 25, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 25, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 25, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Mar 25, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Depolymerase, capsule K1-specific
B: Depolymerase, capsule K1-specific
C: Depolymerase, capsule K1-specific


Theoretical massNumber of molelcules
Total (without water)302,9883
Polymers302,9883
Non-polymers00
Water00
1


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

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Components

#1: Protein Depolymerase, capsule K1-specific / Probable tail fiber protein


Mass: 100996.148 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella phage K64-1 (virus) / Gene: S2-4 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0A8J8T2
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

ComponentName: Depolymerase S2-4 homotrimer / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.3 MDa / Experimental value: NO
Source (natural)Organism: Klebsiella phage K64-1 (virus)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Details of virusEmpty: NO / Enveloped: NO / Isolate: OTHER / Type: VIRION
Buffer solutionpH: 7.5
SpecimenConc.: 1 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: 1200 nm / Cs: 2.7 mm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.18 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 4015926 / Symmetry type: POINT
RefinementResolution: 2.18→273.92 Å / SU ML: 0.11 / σ(F): 0.07 / Phase error: 45.14 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.322 1993 0.05 %
Rwork0.3125 --
obs0.3125 4150106 99.88 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00815705
ELECTRON MICROSCOPYf_angle_d0.95821333
ELECTRON MICROSCOPYf_dihedral_angle_d8.432169
ELECTRON MICROSCOPYf_chiral_restr0.062481
ELECTRON MICROSCOPYf_plane_restr0.0062748
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.18-2.230.60631440.6713294557ELECTRON MICROSCOPY99
2.23-2.290.59231440.5509295860ELECTRON MICROSCOPY100
2.29-2.360.51441430.5404297206ELECTRON MICROSCOPY100
2.36-2.440.55681560.5203295371ELECTRON MICROSCOPY100
2.44-2.530.54511320.4959296957ELECTRON MICROSCOPY100
2.53-2.630.48951200.4523296559ELECTRON MICROSCOPY100
2.63-2.750.42151670.4202296748ELECTRON MICROSCOPY100
2.75-2.890.41191440.3791296109ELECTRON MICROSCOPY100
2.89-3.070.38221200.346296587ELECTRON MICROSCOPY100
3.07-3.310.27241680.3113297265ELECTRON MICROSCOPY100
3.31-3.640.30611440.2625296106ELECTRON MICROSCOPY100
3.64-4.170.21631440.2362296546ELECTRON MICROSCOPY100
4.17-5.250.22441200.1975296587ELECTRON MICROSCOPY100
5.26-273.920.21391470.2036295655ELECTRON MICROSCOPY100

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