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- PDB-9e5e: Escherichia coli DyP peroxidase-loaded encapsulin shell -

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Basic information

Entry
Database: PDB / ID: 9e5e
TitleEscherichia coli DyP peroxidase-loaded encapsulin shell
Components
  • Bacteriocin
  • DyP peroxidase
KeywordsVIRUS LIKE PARTICLE / Dye-decolorizing peroxidase / peroxidase / DyP / encapsulin
Function / homologyType 1 encapsulin shell protein / Encapsulating protein for peroxidase / : / encapsulin nanocompartment / Bacteriocin
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.17 Å
AuthorsAndreas, M.P. / Ubilla, N.C. / Giessen, T.W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: bioRxiv / Year: 2024
Title: Structural and biochemical characterization of a widespread enterobacterial peroxidase encapsulin.
Authors: Natalia C Ubilla-Rodriguez / Michael P Andreas / Tobias W Giessen
Abstract: Encapsulins are self-assembling protein compartments found in prokaryotes and specifically encapsulate dedicated cargo enzymes. The most abundant encapsulin cargo class are Dye-decolorizing ...Encapsulins are self-assembling protein compartments found in prokaryotes and specifically encapsulate dedicated cargo enzymes. The most abundant encapsulin cargo class are Dye-decolorizing Peroxidases (DyPs). It has been previously suggested that DyP encapsulins are involved in oxidative stress resistance and bacterial pathogenicity due to DyPs' inherent ability to reduce and detoxify hydrogen peroxide while oxidizing a broad range of organic co-substrates. Here, we report the structural and biochemical analysis of a DyP encapsulin widely found across enterobacteria. Using bioinformatic approaches, we show that this DyP encapsulin is encoded by a conserved transposon-associated operon, enriched in enterobacterial pathogens. Through low pH and peroxide exposure experiments, we highlight the stability of this DyP encapsulin under harsh conditions and show that DyP catalytic activity is highest at low pH. We determine the structure of the DyP-loaded shell and free DyP via cryo-electron microscopy, revealing the structural basis for DyP cargo loading and peroxide preference. Our work lays the foundation to further explore the substrate range and physiological functions of enterobacterial DyP encapsulins.
History
DepositionOct 28, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 19, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Bacteriocin
B: DyP peroxidase


Theoretical massNumber of molelcules
Total (without water)67,3782
Polymers67,3782
Non-polymers00
Water00
1
A: Bacteriocin
B: DyP peroxidase
x 60


Theoretical massNumber of molelcules
Total (without water)4,042,660120
Polymers4,042,660120
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
2


  • Idetical with deposited unit
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
A: Bacteriocin
B: DyP peroxidase
x 5


  • icosahedral pentamer
  • 337 kDa, 10 polymers
Theoretical massNumber of molelcules
Total (without water)336,88810
Polymers336,88810
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: Bacteriocin
B: DyP peroxidase
x 6


  • icosahedral 23 hexamer
  • 404 kDa, 12 polymers
Theoretical massNumber of molelcules
Total (without water)404,26612
Polymers404,26612
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein Bacteriocin


Mass: 28846.316 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: KTE40
Gene: FPI65_30875, FWK02_13915, GP965_08895, GP975_08820, GP979_05485, GQM21_27145, GRW05_08220, KQO22_005109
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A3L1NQK1
#2: Protein DyP peroxidase


Mass: 38531.352 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Residues 1-338 and 349-351 are not resolved. Residues 339-348 are the DyP-peroxidase targeting peptide.
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: KTE40 / Production host: Escherichia coli BL21(DE3) (bacteria)
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: Escherichia coli KTE40 DyP peroxidase-loaded encapsulin shell
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli) / Strain: KTE40
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5 / Details: 150 mM NaCl, 20 mM Tris pH 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMTRIS(HYDROXYETHYL)AMINOMETHANEC7H17NO31
SpecimenConc.: 2.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grids were glow discharged for 60 seconds at 5 mA under vacuum.
Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295.15 K
Details: Grids were frozen with a blot force of 20, blot time of 4 seconds, 100% humidity, and chamber temperature of 22 degrees C.

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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: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 39.91 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2065
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCv.4.1.2particle selectiontemplate picker
2SerialEMimage acquisition
4cryoSPARCV.4.1.2CTF correctionpatch CTF estimation
7UCSF ChimeraX1.8model fitting
9cryoSPARCv.4.1.2initial Euler assignment
10cryoSPARCv.4.1.2final Euler assignment
12cryoSPARCv.4.1.23D reconstructionhomogeneous refinement
13Coot0.9.8model refinement
14PHENIX1.2.1-4487model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 292965
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 2.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 286236
Details: Homogeneous refinement was used against an ab-initio map with I symmetry, per-particle defocus optimization enabled, per-group CTF parameter optimization enabled, and Ewald sphere correction ...Details: Homogeneous refinement was used against an ab-initio map with I symmetry, per-particle defocus optimization enabled, per-group CTF parameter optimization enabled, and Ewald sphere correction enabled using a positive curvature sign.
Symmetry type: POINT
Atomic model buildingB value: 85.5 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation coefficient
Details: The AlphaFold model was placed using ChimeraX, followed by iterative manual refinement in Coot followed by Phenix real space refinements.
Atomic model building
ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
11A0A3L1NQK11AlphaFoldin silico model
21A0A747AN602AlphaFoldin silico model

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