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- PDB-9b9i: Myxococcus xanthus EncA encapsulin engineered pore mutant with T=... -

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Basic information

Entry
Database: PDB / ID: 9b9i
TitleMyxococcus xanthus EncA encapsulin engineered pore mutant with T=1 icosahedral symmetry
ComponentsType 1 encapsulin shell protein EncA
KeywordsVIRUS LIKE PARTICLE / encapsulin / nanocompartment / pore mutant
Function / homology: / Type 1 encapsulin shell protein / Encapsulating protein for peroxidase / encapsulin nanocompartment / iron ion transport / intracellular iron ion homeostasis / Type 1 encapsulin shell protein EncA
Function and homology information
Biological speciesMyxococcus xanthus DK 1622 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.86 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 2, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 18, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 25, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Type 1 encapsulin shell protein EncA


Theoretical massNumber of molelcules
Total (without water)30,9011
Polymers30,9011
Non-polymers00
Water00
1
A: Type 1 encapsulin shell protein EncA
x 60


Theoretical massNumber of molelcules
Total (without water)1,854,06160
Polymers1,854,06160
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
x 5


  • icosahedral pentamer
  • 155 kDa, 5 polymers
Theoretical massNumber of molelcules
Total (without water)154,5055
Polymers154,5055
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: Type 1 encapsulin shell protein EncA
x 6


  • icosahedral 23 hexamer
  • 185 kDa, 6 polymers
Theoretical massNumber of molelcules
Total (without water)185,4066
Polymers185,4066
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 / Myxococcus xanthus EncA encapsulin engineered pore mutant with T=1 icosahedral symmetry


Mass: 30901.014 Da / Num. of mol.: 1 / Fragment: I203G, Y204G, del(205-210)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Myxococcus xanthus DK 1622 (bacteria) / Gene: encA, MXAN_3556 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q1D6H4

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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

ComponentName: Myxococcus xanthus EncA encapsulin engineered pore mutant with T=1 icosahedral symmetry
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.9 MDa / Experimental value: YES
Source (natural)Organism: Myxococcus xanthus DK 1622 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Details of virusEmpty: YES / Enveloped: NO / Isolate: STRAIN / Type: VIRUS-LIKE PARTICLE
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.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: The grid was glow discharged at 5 mA for 60 seconds under vacuum.
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: 4 sec. / Electron dose: 39.18 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 711
Image scansWidth: 3710 / Height: 3838 / Movie frames/image: 20

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC3.10.0particle selectionTemplate Picker
2Leginonimage acquisition
4cryoSPARC3.10.0CTF correctionPatch CTF
7UCSF ChimeraX1.2.5model fitting
9cryoSPARC3.10.0initial Euler assignment
10cryoSPARC3.10.0final Euler assignment
12cryoSPARC3.10.03D reconstructionHomogeneous refinement
19PHENIXv1.20.1-4487-000model refinement
20Cootv0.9.8.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 66813
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 61808
Details: CryoSPARC homogeneous refinement was performed with I symmetry, per-particle defocus optimization, per-group CTF-parameterization, and Ewald sphere correction enabled.
Symmetry type: POINT
Atomic model buildingB value: 126.4 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation coefficient
Details: ChimeraX v1.2.5 was first used to place the starting model (PDB: 7S21) 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: 7S21) 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: 7S21

7s21


Accession code: 7S21 / Source name: PDB / Type: experimental model

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