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- PDB-8u50: Klebsiella pneumoniae DyP peroxidase-loaded encapsulin shell -

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Basic information

Entry
Database: PDB / ID: 8u50
TitleKlebsiella pneumoniae DyP peroxidase-loaded encapsulin shell
ComponentsKlebsiella pneumoniae family 1 encapsulin shell
KeywordsVIRUS LIKE PARTICLE / Encapsulin / nanocompartment / DyP peroxidase
Biological speciesKlebsiella pneumoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.52 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: Klebsiella pneumoniae family 1 encapsulin shell


Theoretical massNumber of molelcules
Total (without water)28,8411
Polymers28,8411
Non-polymers00
Water0
1
A: Klebsiella pneumoniae family 1 encapsulin shell
x 60


Theoretical massNumber of molelcules
Total (without water)1,730,46860
Polymers1,730,46860
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: Klebsiella pneumoniae family 1 encapsulin shell
x 5


  • icosahedral pentamer
  • 144 kDa, 5 polymers
Theoretical massNumber of molelcules
Total (without water)144,2065
Polymers144,2065
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: Klebsiella pneumoniae family 1 encapsulin shell
x 6


  • icosahedral 23 hexamer
  • 173 kDa, 6 polymers
Theoretical massNumber of molelcules
Total (without water)173,0476
Polymers173,0476
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 Klebsiella pneumoniae family 1 encapsulin shell


Mass: 28841.129 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: GBV82_RS22365 / Production host: Escherichia coli BL21(DE3) (bacteria)

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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
1Klebsiella pneumoniae DyP peroxidase-loaded encapsulin shellCOMPLEXall0RECOMBINANT
2Klebsiella pneumoniae family 1 encapsulin shellCOMPLEXall1RECOMBINANT
3Klebsiella pneumoniae encapsulated DyP peroxidaseCOMPLEX2RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Klebsiella pneumoniae (bacteria)573
32Klebsiella pneumoniae (bacteria)573
43Klebsiella pneumoniae (bacteria)573
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli BL21(DE3) (bacteria)469008
32Escherichia coli BL21(DE3) (bacteria)469008
43Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7.5 / Details: 20 mM Tris pH 7.5, 150 mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
120 mmTrisC4H12NO31
2150 mmSodium chlorideNaClSodium chloride1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Glow discharged for 60 seconds at 5 mA / 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: Blot force: 20 Blot time: 4 seconds

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 45000 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: 5 sec. / Electron dose: 41.67 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1183
Image scansWidth: 3710 / Height: 3838 / Movie frames/image: 25

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.2.1particle selectionTemplate Picker
2Leginonimage acquisition
4cryoSPARC4.2.1CTF correctionPatch CTF Estimation
7UCSF ChimeraX1.2.5model fitting
8Coot0.9.8model fitting
10PHENIX1.20.1-4487-000model refinement
11cryoSPARC4.2.1initial Euler assignment
12cryoSPARC4.2.1final Euler assignment
14cryoSPARC4.2.13D reconstructionHomogenous refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 75967
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 2.52 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 64969 / Symmetry type: POINT
Atomic model buildingB value: 95.2 / 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 buildingPDB-ID: 7BOJ

7boj


Accession code: 7BOJ / Source name: PDB / Type: experimental model

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