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- EMDB-41904: Klebsiella pneumoniae encapsulin-associated DyP peroxidase -

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Basic information

Entry
Database: EMDB / ID: EMD-41904
TitleKlebsiella pneumoniae encapsulin-associated DyP peroxidase
Map dataKlebsiella pneumoniae encapsulin-associated DyP peroxidase
Sample
  • Complex: Klebsiella pneumoniae family 1 encapsulin-associated DyP peroxidase
    • Protein or peptide: Family 1 encapsulin-associated DyP peroxidase
  • Ligand: Mesoheme
KeywordsDyP peroxidase / encapsulin / OXIDOREDUCTASE
Biological speciesKlebsiella pneumoniae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.39 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_41904.png

Downloads & links

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Map

FileDownload / File: emd_41904.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationKlebsiella pneumoniae encapsulin-associated DyP peroxidase
Voxel sizeX=Y=Z: 0.832 Å
Density
Contour LevelBy AUTHOR: 0.06
Minimum - Maximum-0.3818379 - 0.6607309
Average (Standard dev.)0.00031092373 (±0.020920508)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 212.992 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map B

Fileemd_41904_half_map_1.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_41904_half_map_2.map
AnnotationHalf Map A
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 family 1 encapsulin-associated DyP peroxidase

EntireName: Klebsiella pneumoniae family 1 encapsulin-associated DyP peroxidase
Components
  • Complex: Klebsiella pneumoniae family 1 encapsulin-associated DyP peroxidase
    • Protein or peptide: Family 1 encapsulin-associated DyP peroxidase
  • Ligand: Mesoheme

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Supramolecule #1: Klebsiella pneumoniae family 1 encapsulin-associated DyP peroxidase

SupramoleculeName: Klebsiella pneumoniae family 1 encapsulin-associated DyP peroxidase
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 240 KDa

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Macromolecule #1: Family 1 encapsulin-associated DyP peroxidase

MacromoleculeName: Family 1 encapsulin-associated DyP peroxidase / type: protein_or_peptide / ID: 1
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.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 40.691395 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MACPISQSVS QPVDERLTRA AIFLVVTINP GKAAEVAVRT LCGTLSSLVR GVGFRILDGG LSCVMGVSSG GWERLFGDEK PEYLHVFQE INGVHHAPST PGDLLFHIRA ARMDLCFELA SRILSDLGSS VCVVDSVQGF RYFDDRDLLG FVDGTENPVA Q AAVDATLI ...String:
MACPISQSVS QPVDERLTRA AIFLVVTINP GKAAEVAVRT LCGTLSSLVR GVGFRILDGG LSCVMGVSSG GWERLFGDEK PEYLHVFQE INGVHHAPST PGDLLFHIRA ARMDLCFELA SRILSDLGSS VCVVDSVQGF RYFDDRDLLG FVDGTENPVA Q AAVDATLI GEEDHTFSGG SYVIVQKYLH DLDKWNAIPV EQQEKIIGRE KLSDIELKDA DKPSYAHNVL TSIEEDGEDV DI LRDNMPF GDPGKGEFGT YFIGYSRKPA RIERMLENMF VGNPPGNYDR ILDVSRAITG TLFFIPTVSF LDSVEPQPSV SQQ TDDAKY IYDSPGTKGE SGSLNIGSLK KEVQDEENLY FQGGSGGHHH HHHH

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Macromolecule #2: Mesoheme

MacromoleculeName: Mesoheme / type: ligand / ID: 2 / Number of copies: 1 / Formula: MH0
Molecular weightTheoretical: 620.519 Da
Chemical component information

ChemComp-MH0:
Mesoheme

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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

Concentration0.45 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 R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Details: 60 seconds at 5 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV / Details: Blot force: 5 Blot time: 2 seconds.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 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: 105000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 2468 / Average exposure time: 1.89747 sec. / Average electron dose: 50.02 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2603020
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.0+231114)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.0+231114)
Final reconstructionApplied symmetry - Point group: D3 (2x3 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.39 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.0+231114) / Software - details: Local Refinement
Details: Unmasked local refinement was performed with D3 symmetry imposed.
Number images used: 431317
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6-f1


Chain - Source name: Other / Chain - Initial model type: in silico model
Details: An AlphaFill model containing bound heme was used as starting 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: 107.7 / Target criteria: Cross-correlation coefficient
Output model

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

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