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- EMDB-41905: Klebsiella pneumoniae DyP peroxidase-loaded encapsulin shell -

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Basic information

Entry
Database: EMDB / ID: EMD-41905
TitleKlebsiella pneumoniae DyP peroxidase-loaded encapsulin shell
Map dataKlebsiella pneumoniae DyP peroxidase-loaded encapsulin shell
Sample
  • Complex: Klebsiella pneumoniae DyP peroxidase-loaded encapsulin shell
    • Complex: Klebsiella pneumoniae family 1 encapsulin shell
      • Complex: Klebsiella pneumoniae encapsulated DyP peroxidase
      • Protein or peptide: Klebsiella pneumoniae family 1 encapsulin shell
KeywordsEncapsulin / nanocompartment / DyP peroxidase / VIRUS LIKE PARTICLE
Biological speciesKlebsiella pneumoniae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.52 Å
AuthorsAndreas MP / Jones JA / Giessen TW
Funding support United States, 1 items
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, 2023-
Header (metadata) releaseApr 24, 2024-
Map releaseApr 24, 2024-
UpdateApr 24, 2024-
Current statusApr 24, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41905.png

Downloads & links

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Map

FileDownload / File: emd_41905.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationKlebsiella pneumoniae DyP peroxidase-loaded encapsulin shell
Voxel sizeX=Y=Z: 0.91 Å
Density
Contour LevelBy AUTHOR: 0.28
Minimum - Maximum-0.7253012 - 1.2741572
Average (Standard dev.)0.0039213775 (±0.07172879)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 349.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map 1

Fileemd_41905_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_41905_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Klebsiella pneumoniae DyP peroxidase-loaded encapsulin shell

EntireName: Klebsiella pneumoniae DyP peroxidase-loaded encapsulin shell
Components
  • Complex: Klebsiella pneumoniae DyP peroxidase-loaded encapsulin shell
    • Complex: Klebsiella pneumoniae family 1 encapsulin shell
      • Complex: Klebsiella pneumoniae encapsulated DyP peroxidase
      • Protein or peptide: Klebsiella pneumoniae family 1 encapsulin shell

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Supramolecule #1: Klebsiella pneumoniae DyP peroxidase-loaded encapsulin shell

SupramoleculeName: Klebsiella pneumoniae DyP peroxidase-loaded encapsulin shell
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Klebsiella pneumoniae (bacteria)

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Supramolecule #2: Klebsiella pneumoniae family 1 encapsulin shell

SupramoleculeName: Klebsiella pneumoniae family 1 encapsulin shell / type: complex / ID: 2 / Parent: 1 / Macromolecule list: all
Source (natural)Organism: Klebsiella pneumoniae (bacteria)

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Supramolecule #3: Klebsiella pneumoniae encapsulated DyP peroxidase

SupramoleculeName: Klebsiella pneumoniae encapsulated DyP peroxidase / type: complex / ID: 3 / Parent: 2
Source (natural)Organism: Klebsiella pneumoniae (bacteria)

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Macromolecule #1: Klebsiella pneumoniae family 1 encapsulin shell

MacromoleculeName: Klebsiella pneumoniae family 1 encapsulin shell / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 28.841129 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNNLHRELAP VSDAAWEQIE EEASRTLKRF LAARRVVDVS DPQGPAFSAV GTGHVTRLEG PGDSVGAVKR QSQPVVEFRV PFILTRQAI DDVERGSQDS DWSPLKEAAR KIAGAEDRAV FDGYAAAGIG GIRPQSSNSP LTLPVAASGY PDVIARALDQ L RVAGVNGP ...String:
MNNLHRELAP VSDAAWEQIE EEASRTLKRF LAARRVVDVS DPQGPAFSAV GTGHVTRLEG PGDSVGAVKR QSQPVVEFRV PFILTRQAI DDVERGSQDS DWSPLKEAAR KIAGAEDRAV FDGYAAAGIG GIRPQSSNSP LTLPVAASGY PDVIARALDQ L RVAGVNGP YHLVLGENAY TLITSGNEDG YPVLQHIHRL IDGEIVWAPA IEGGVLLSTR GGDFAMDIGQ DISIGYLSHT AT HVELYLQ ESFTFRTLTS EAVVSLLPSE D

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mmC4H12NO3Tris
150.0 mmNaClSodium chlorideSodium chloride

Details: 20 mM Tris pH 7.5, 150 mM NaCl
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Details: Glow discharged for 60 seconds at 5 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV / Details: Blot force: 20 Blot time: 4 seconds.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 45000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Number grids imaged: 1 / Number real images: 1183 / Average exposure time: 5.0 sec. / Average electron dose: 41.67 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 75967
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 4.2.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 4.2.1)
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 2.52 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Software - details: Homogenous refinement / Number images used: 64969
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7boj


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsInitial 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.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 95.2 / Target criteria: Cross-correlation coefficient
Output model

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

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