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- PDB-9e4r: Escherichia coli encapsulin-associated DyP peroxidase -

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ID or keywords:

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Basic information

Entry
Database: PDB / ID: 9e4r
TitleEscherichia coli encapsulin-associated DyP peroxidase
ComponentsDyp-type peroxidase
KeywordsOXIDOREDUCTASE / Dye-decolorizing peroxidase / peroxidase / DyP / encapsulin
Function / homology
Function and homology information


peroxidase activity / heme binding / metal ion binding / cytosol
Similarity search - Function
: / : / Dyp-type peroxidase, C-terminal / Dyp-type peroxidase, N-terminal / DyP-type peroxidase family. / Dyp-type peroxidase / Dimeric alpha-beta barrel
Similarity search - Domain/homology
PROTOPORPHYRIN IX CONTAINING FE / Dyp-type peroxidase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.31 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 25, 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

Downloads & links

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Assembly

Deposited unit
A: Dyp-type peroxidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)41,2612
Polymers40,6451
Non-polymers6161
Water00
1
A: Dyp-type peroxidase
hetero molecules
x 6


Theoretical massNumber of molelcules
Total (without water)247,56612
Polymers243,8676
Non-polymers3,6996
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5
2


  • Idetical with deposited unit
  • point asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: D3 (2x3 fold dihedral))

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Components

#1: Protein Dyp-type peroxidase


Mass: 40644.516 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: KTE40 / Gene: FPI65_30880, FWK02_13910 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A5C9AJY8
#2: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: C34H32FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
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 encapsulin-associated DyP peroxidase
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli) / Strain: KTE40
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Details: Sample was prepared in 20 mM Tris pH 7.5, 150 mM NaCl.
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMtris(hydroxymethyl)aminomethaneC4H11NO31
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was frozen on two grids, one at 0.5 mg/mL and the other at 0.25 mg/mL.
Specimen supportDetails: Grids were glow discharged for 60 seconds at 5 mA in 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 K
Details: Grids were frozen with the following parameters: blot force 20, blot time 4 seconds, humidty 100 percent, 22 degrees C.

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
EM imaging

Accelerating voltage: 200 kV / Electron source: FIELD EMISSION GUN / Illumination mode: FLOOD BEAM / Mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1800 nm / Nominal defocus min: 1000 nm / Nominal magnification: 45000 X / Specimen-ID: 1

IDCryogenModel
1NITROGENFEI TALOS ARCTICA
2TFS GLACIOS
Image recording

Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1

IDImaging-IDAverage exposure time (sec.)Electron dose (e/Å2)Num. of real images
11647.84123
22446.24206
Image scans
WidthHeightMovie frames/imageIDImage recording-IDEntry-ID
3710383830119E4R
3710383820229E4R

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Processing

EM software
IDNameVersionCategoryImaging-IDDetails
1cryoSPARC4.5.1particle selection
2Leginonimage acquisition1
4cryoSPARC4.5.1CTF correction
7UCSF ChimeraX1.8model fitting
9Leginonimage acquisition2
10PHENIX1.2.1-4887model refinementreal space refinement
11Coot0.9.8.1model refinement
12cryoSPARC4.5.1initial Euler assignment
13cryoSPARC4.5.1final Euler assignment
15cryoSPARC4.5.13D reconstructionLocal Refinement
Image processingDetails: Particles were picked from micrographs collected on two different grids using TFS Glacios and FEI Talos Arctica cryogenic electron microscopes.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 115178
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 3.31 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 10490
Details: Local refinement with D3 symmetry and force re-do GS-split applied.
Symmetry type: POINT
Atomic model buildingB value: 105.9 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation coefficient
Atomic model buildingAccession code: A0A747AN60 / Source name: AlphaFold / Type: in silico model

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