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- EMDB-44427: Cargo-loaded Myxococcus xanthus EncA encapsulin engineered pore m... -

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Basic information

Entry
Database: EMDB / ID: EMD-44427
TitleCargo-loaded Myxococcus xanthus EncA encapsulin engineered pore mutant with T=4 icosahedral symmetry
Map data
Sample
  • Complex: Cargo-loaded Myxococcus xanthus EncA encapsulin engineered pore mutant with T=4 icosahedral symmetry
    • Complex: Myxococcus xanthus EncA encapsulin engineered pore mutant with T=4 icosahedral symmetry
      • Complex: SNAP-tag-targeting peptide cargo protein
        • Protein or peptide: Encapsulin nanocompartment cargo protein EncC
      • Protein or peptide: Type 1 encapsulin shell protein EncA
Keywordsencapsulin / nanocompartment / pore mutant / VIRUS LIKE PARTICLE
Function / homology
Function and homology information


encapsulin nanocompartment / iron ion transport / intracellular iron ion homeostasis / metal ion binding
Similarity search - Function
: / EncFtn-like / Type 1 encapsulin shell protein / Encapsulating protein for peroxidase / : / Ferritin-like superfamily
Similarity search - Domain/homology
Encapsulin nanocompartment cargo protein EncC / Type 1 encapsulin shell protein EncA
Similarity search - Component
Biological speciesMyxococcus xanthus DK 1622 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.46 Å
AuthorsAndreas MP / Kwon S / Giessen TW
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM133325-05 United States
CitationJournal: ACS Nano / Year: 2024
Title: Pore Engineering as a General Strategy to Improve Protein-Based Enzyme Nanoreactor Performance.
Authors: Seokmu Kwon / Michael P Andreas / Tobias W Giessen /
Abstract: Enzyme nanoreactors are nanoscale compartments consisting of encapsulated enzymes and a selectively permeable barrier. Sequestration and colocalization of enzymes can increase catalytic activity, ...Enzyme nanoreactors are nanoscale compartments consisting of encapsulated enzymes and a selectively permeable barrier. Sequestration and colocalization of enzymes can increase catalytic activity, stability, and longevity, highly desirable features for many biotechnological and biomedical applications of enzyme catalysts. One promising strategy to construct enzyme nanoreactors is to repurpose protein nanocages found in nature. However, protein-based enzyme nanoreactors often exhibit decreased catalytic activity, partially caused by a mismatch of protein shell selectivity and the substrate requirements of encapsulated enzymes. No broadly applicable and modular protein-based nanoreactor platform is currently available. Here, we introduce a pore-engineered universal enzyme nanoreactor platform based on encapsulins-microbial self-assembling protein nanocompartments with programmable and selective enzyme packaging capabilities. We structurally characterize our protein shell designs via cryo-electron microscopy and highlight their polymorphic nature. Through fluorescence polarization assays, we show their improved molecular flux behavior and highlight their expanded substrate range via a number of proof-of-concept enzyme nanoreactor designs. This work lays the foundation for utilizing our encapsulin-based nanoreactor platform for diverse future biotechnological and biomedical applications.
History
DepositionApr 8, 2024-
Header (metadata) releaseOct 30, 2024-
Map releaseOct 30, 2024-
UpdateOct 30, 2024-
Current statusOct 30, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44427.png

Downloads & links

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Map

FileDownload / File: emd_44427.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
1.16 Å/pix.
x 440 pix.
= 509.6 Å		1.16 Å/pix.
x 440 pix.
= 509.6 Å		1.16 Å/pix.
x 440 pix.
= 509.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.15818 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-0.27947837 - 0.6740436
Average (Standard dev.)0.008247828 (±0.054064833)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 509.60004 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_44427_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_44427_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cargo-loaded Myxococcus xanthus EncA encapsulin engineered pore m...

EntireName: Cargo-loaded Myxococcus xanthus EncA encapsulin engineered pore mutant with T=4 icosahedral symmetry
Components
  • Complex: Cargo-loaded Myxococcus xanthus EncA encapsulin engineered pore mutant with T=4 icosahedral symmetry
    • Complex: Myxococcus xanthus EncA encapsulin engineered pore mutant with T=4 icosahedral symmetry
      • Complex: SNAP-tag-targeting peptide cargo protein
        • Protein or peptide: Encapsulin nanocompartment cargo protein EncC
      • Protein or peptide: Type 1 encapsulin shell protein EncA

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Supramolecule #1: Cargo-loaded Myxococcus xanthus EncA encapsulin engineered pore m...

SupramoleculeName: Cargo-loaded Myxococcus xanthus EncA encapsulin engineered pore mutant with T=4 icosahedral symmetry
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Myxococcus xanthus DK 1622 (bacteria)
Molecular weightTheoretical: 7.4 MDa

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Supramolecule #2: Myxococcus xanthus EncA encapsulin engineered pore mutant with T=...

SupramoleculeName: Myxococcus xanthus EncA encapsulin engineered pore mutant with T=4 icosahedral symmetry
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Myxococcus xanthus DK 1622 (bacteria)

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Supramolecule #3: SNAP-tag-targeting peptide cargo protein

SupramoleculeName: SNAP-tag-targeting peptide cargo protein / type: complex / ID: 3 / Parent: 2 / Macromolecule list: #2
Source (natural)Organism: Myxococcus xanthus DK 1622 (bacteria)

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Macromolecule #1: Type 1 encapsulin shell protein EncA

MacromoleculeName: Type 1 encapsulin shell protein EncA / type: protein_or_peptide / ID: 1 / Details: del(205-210) / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Myxococcus xanthus DK 1622 (bacteria)
Molecular weightTheoretical: 30.901014 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MPDFLGHAEN PLREEEWARL NETVIQVARR SLVGRRILDI YGPLGAGVQT VPYDEFQGVS PGAVDIVGEQ ETAMVFTDAR KFKTIPIIY KDFLLHWRDI EAARTHNMPL DVSAAAGAAA LCAQQEDELI FYGDARLGYE GLMTANGRLT VPLGDWTSPG G GFQAIVEA ...String:
MPDFLGHAEN PLREEEWARL NETVIQVARR SLVGRRILDI YGPLGAGVQT VPYDEFQGVS PGAVDIVGEQ ETAMVFTDAR KFKTIPIIY KDFLLHWRDI EAARTHNMPL DVSAAAGAAA LCAQQEDELI FYGDARLGYE GLMTANGRLT VPLGDWTSPG G GFQAIVEA TRKLNEQGHF GPYAVVLSPR LYSQLHRGGE IETIRQLASD GVYQSNRLRG ESGVVVSTGR ENMDLAVSMD MV AAYLGAS RMNHPFRVLE ALLLRIKHPD AICTLEGAGA TERR

UniProtKB: Type 1 encapsulin shell protein EncA

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Macromolecule #2: Encapsulin nanocompartment cargo protein EncC

MacromoleculeName: Encapsulin nanocompartment cargo protein EncC / type: protein_or_peptide / ID: 2
Details: C-terminal targeting peptide of a SNAP-Tag-targeting peptide cargo protein.
Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Myxococcus xanthus DK 1622 (bacteria)
Molecular weightTheoretical: 1.415685 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
PEKRLTVGSL RR

UniProtKB: Encapsulin nanocompartment cargo protein EncC

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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

Concentration4.1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMC4H11NO3tris(hydroxymethyl)aminomethane

Details: 150 mM NaCl, 20 mM Tris pH 7.5
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 discharge was performed under vacuum at 5 mA for 60 seconds.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
Details: Blot force: 20 Blot time: 4 seconds Wait time: 0 seconds.

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Electron microscopy

MicroscopeFEI TECNAI ARCTICA
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: 706 / Average exposure time: 6.0 sec. / Average electron dose: 39.56 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 45000
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 37600
Startup modelType of model: INSILICO MODEL / Details: Ab-initio model
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.10.0) / Software - details: Homogeneous Refinement
Details: Homogeneous refinement was performed against the intial ab-initio map using I symmetry, per-particle defocus optimization, per-group CTF parameterization, and Ewald sphere correction.
Number images used: 4386
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.10.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.10.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7s4q


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsChimeraX v1.2.5 was first used to place the starting model (PDB: 7S4Q) in the cryo-EM map by using the fit in map command. The model was then manually refined using Coot v 0.9.8.1, followed by iterative real-space refinements in Phenix v1.20.1-4487-000. BioMT operators were identified from the cryo-EM map using map_symmetry command in Phenix then applied to the model using the apply_ncs command to assemble the complete shell. Real-space refinement was repeated in Phenix with NCS constraints applied. The BioMT operators were then identified using find_ncs command in Phenix and applied to the header of a protomer of the NCS-refined model.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 60.6 / Target criteria: cross-correlation coefficient
Output model

PDB-9bc8:
Cargo-loaded Myxococcus xanthus EncA encapsulin engineered pore mutant with T=4 icosahedral symmetry

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