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- PDB-8u4z: Klebsiella pneumoniae encapsulin-associated DyP peroxidase -

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Basic information

Entry
Database: PDB / ID: 8u4z
TitleKlebsiella pneumoniae encapsulin-associated DyP peroxidase
ComponentsFamily 1 encapsulin-associated DyP peroxidase
KeywordsOXIDOREDUCTASE / DyP peroxidase / encapsulin
Function / homologyMesoheme
Function and homology information
Biological speciesKlebsiella pneumoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.39 Å
AuthorsAndreas, M.P. / Jones, J.A. / Giessen, T.W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R35GM133325-04 United States
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis for peroxidase encapsulation inside the encapsulin from the Gram-negative pathogen Klebsiella pneumoniae.
Authors: Jesse A Jones / Michael P Andreas / Tobias W Giessen /
Abstract: Encapsulins are self-assembling protein nanocompartments capable of selectively encapsulating dedicated cargo proteins, including enzymes involved in iron storage, sulfur metabolism, and stress ...Encapsulins are self-assembling protein nanocompartments capable of selectively encapsulating dedicated cargo proteins, including enzymes involved in iron storage, sulfur metabolism, and stress resistance. They represent a unique compartmentalization strategy used by many pathogens to facilitate specialized metabolic capabilities. Encapsulation is mediated by specific cargo protein motifs known as targeting peptides (TPs), though the structural basis for encapsulation of the largest encapsulin cargo class, dye-decolorizing peroxidases (DyPs), is currently unknown. Here, we characterize a DyP-containing encapsulin from the enterobacterial pathogen Klebsiella pneumoniae. By combining cryo-electron microscopy with TP and TP-binding site mutagenesis, we elucidate the molecular basis for cargo encapsulation. TP binding is mediated by cooperative hydrophobic and ionic interactions as well as shape complementarity. Our results expand the molecular understanding of enzyme encapsulation inside protein nanocompartments and lay the foundation for rationally modulating encapsulin cargo loading for biomedical and biotechnological applications.
History
DepositionSep 11, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 24, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Family 1 encapsulin-associated DyP peroxidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)41,3122
Polymers40,6911
Non-polymers6211
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
SymmetryPoint symmetry: (Schoenflies symbol: D3 (2x3 fold dihedral))

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Components

#1: Protein Family 1 encapsulin-associated DyP peroxidase


Mass: 40691.395 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Residues 1-352 are K.p. DyP peroxidase (NCBI: WP_193221875.1). Residues 1-2 and 316-352 are disordered. Residues 353-370 are a TEV protease site, linker, and HIS-tag that are disordered in the structure.
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: GBV82_RS22370 / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Chemical ChemComp-MH0 / Mesoheme


Mass: 620.519 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C34H36FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: Klebsiella pneumoniae family 1 encapsulin-associated DyP peroxidase
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.24 MDa / Experimental value: YES
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5 / Details: 20 mM Tris pH 7.5, 150 mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
120 mmTrisC4H12NO31
2150 mmSodium chlorideNaCl1
SpecimenConc.: 0.45 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 60 seconds at 5 mA / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K / Details: Blot force: 5 Blot time: 2 seconds

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN 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: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 1.89747 sec. / Electron dose: 50.02 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2468
Image scansWidth: 5760 / Height: 4092 / Movie frames/image: 20

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.4.0+231114particle selectionTemplate picker
2SerialEMimage acquisition
4cryoSPARC4.4.0+231114CTF correctionPatch CTF Estimation in cryoSPARC Live
7UCSF ChimeraX1.2.5model fitting
8Coot0.9.8model fitting
10PHENIX1.20.1-4487-000model refinement
11cryoSPARC4.4.0+231114initial Euler assignment
12cryoSPARC4.4.0+231114final Euler assignment
14cryoSPARC4.4.0+2311143D reconstructionLocal Refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2603020
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 2.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 431317
Details: Unmasked local refinement was performed with D3 symmetry imposed.
Symmetry type: POINT
Atomic model buildingB value: 107.7 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: Initial fitting was performed using ChimeraX v1.2.5. The model was then manually refined and mutated using Coot v9.8.1 followed by real-space refinement using Phenix v1.20.1-4487-000.
Atomic model buildingAccession code: AF-A0A3Z8UGY6-F1
Details: An AlphaFill model containing bound heme was used as starting model
Source name: Other / Type: in silico model

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