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- PDB-9bc8: Cargo-loaded Myxococcus xanthus EncA encapsulin engineered pore m... -

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Basic information

Entry
Database: PDB / ID: 9bc8
TitleCargo-loaded Myxococcus xanthus EncA encapsulin engineered pore mutant with T=4 icosahedral symmetry
Components
  • Encapsulin nanocompartment cargo protein EncC
  • Type 1 encapsulin shell protein EncA
KeywordsVIRUS LIKE PARTICLE / encapsulin / nanocompartment / pore mutant
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.46 Å
AuthorsAndreas, M.P. / Kwon, S. / Giessen, T.W.
Funding support United States, 1items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 30, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Type 1 encapsulin shell protein EncA
B: Type 1 encapsulin shell protein EncA
C: Type 1 encapsulin shell protein EncA
D: Type 1 encapsulin shell protein EncA
E: Encapsulin nanocompartment cargo protein EncC
F: Encapsulin nanocompartment cargo protein EncC
G: Encapsulin nanocompartment cargo protein EncC
H: Encapsulin nanocompartment cargo protein EncC


Theoretical massNumber of molelcules
Total (without water)129,2678
Polymers129,2678
Non-polymers00
Water00
1
A: Type 1 encapsulin shell protein EncA
B: Type 1 encapsulin shell protein EncA
C: Type 1 encapsulin shell protein EncA
D: Type 1 encapsulin shell protein EncA
E: Encapsulin nanocompartment cargo protein EncC
F: Encapsulin nanocompartment cargo protein EncC
G: Encapsulin nanocompartment cargo protein EncC
H: Encapsulin nanocompartment cargo protein EncC
x 60


Theoretical massNumber of molelcules
Total (without water)7,756,008480
Polymers7,756,008480
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: Type 1 encapsulin shell protein EncA
B: Type 1 encapsulin shell protein EncA
C: Type 1 encapsulin shell protein EncA
D: Type 1 encapsulin shell protein EncA
E: Encapsulin nanocompartment cargo protein EncC
F: Encapsulin nanocompartment cargo protein EncC
G: Encapsulin nanocompartment cargo protein EncC
H: Encapsulin nanocompartment cargo protein EncC
x 5


  • icosahedral pentamer
  • 646 kDa, 40 polymers
Theoretical massNumber of molelcules
Total (without water)646,33440
Polymers646,33440
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: Type 1 encapsulin shell protein EncA
B: Type 1 encapsulin shell protein EncA
C: Type 1 encapsulin shell protein EncA
D: Type 1 encapsulin shell protein EncA
E: Encapsulin nanocompartment cargo protein EncC
F: Encapsulin nanocompartment cargo protein EncC
G: Encapsulin nanocompartment cargo protein EncC
H: Encapsulin nanocompartment cargo protein EncC
x 6


  • icosahedral 23 hexamer
  • 776 kDa, 48 polymers
Theoretical massNumber of molelcules
Total (without water)775,60148
Polymers775,60148
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
Type 1 encapsulin shell protein EncA / Cargo-loaded Myxococcus xanthus EncA encapsulin engineered pore mutant with T=3 icosahedral symmetry


Mass: 30901.014 Da / Num. of mol.: 4 / Mutation: I203G, Y204G
Source method: isolated from a genetically manipulated source
Details: del(205-210) / Source: (gene. exp.) Myxococcus xanthus DK 1622 (bacteria) / Gene: encA, MXAN_3556 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q1D6H4
#2: Protein/peptide
Encapsulin nanocompartment cargo protein EncC / SNAP-tag-targeting peptide cargo protein


Mass: 1415.685 Da / Num. of mol.: 4
Fragment: C-terminal targeting peptide (UNP residues 119-130)
Source method: isolated from a genetically manipulated source
Details: C-terminal targeting peptide of a SNAP-Tag-targeting peptide cargo protein.
Source: (gene. exp.) Myxococcus xanthus DK 1622 (bacteria) / Gene: encC, MXAN_4464 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q1D3Y8
Has protein modificationN

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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
1Cargo-loaded Myxococcus xanthus EncA encapsulin engineered pore mutant with T=4 icosahedral symmetryCOMPLEXall0RECOMBINANT
2Myxococcus xanthus EncA encapsulin engineered pore mutant with T=4 icosahedral symmetryCOMPLEX#11RECOMBINANT
3SNAP-tag-targeting peptide cargo proteinCOMPLEX#22RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
117.4 MDaYES
217.4 MDaYES
33
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Myxococcus xanthus DK 1622 (bacteria)246197
32Myxococcus xanthus DK 1622 (bacteria)246197
43Myxococcus xanthus DK 1622 (bacteria)246197
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: 150 mM NaCl, 20 mM Tris pH 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMtris(hydroxymethyl)aminomethaneC4H11NO31
SpecimenConc.: 4.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Glow discharge was performed under vacuum at 5 mA for 60 seconds.
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 K
Details: Blot force: 20 Blot time: 4 seconds Wait time: 0 seconds

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 45000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 1000 nm
Image recordingAverage exposure time: 6 sec. / Electron dose: 39.56 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 706
Image scansWidth: 3710 / Height: 3838 / Movie frames/image: 30

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.10.0particle selectionTemplate Picker
2Leginonimage acquisition
4cryoSPARC4.10.0CTF correctionPatch CTF Estimation
7UCSF ChimeraX1.2.5model fitting
9Coot0.9.8.1model refinement
10PHENIX1.20.1-4487-000model refinement
11cryoSPARC4.10.0initial Euler assignment
12cryoSPARC4.10.0final Euler assignment
14cryoSPARC4.10.03D reconstructionHomogeneous Refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 37600
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 4386
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.
Symmetry type: POINT
Atomic model buildingB value: 60.6 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation coefficient
Details: ChimeraX 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 ...Details: ChimeraX 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.
Atomic model buildingPDB-ID: 7S4Q

7s4q


Accession code: 7S4Q / Source name: PDB / Type: experimental model

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