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- PDB-9zw3: Quasibacillus thermotolerans T=4 encapsulin pore mutant variant L... -

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Basic information

Entry
Database: PDB / ID: 9zw3
TitleQuasibacillus thermotolerans T=4 encapsulin pore mutant variant Letter11
ComponentsType 1 encapsulin shell protein
KeywordsVIRUS LIKE PARTICLE / encapsulin / protein nanocompartment
Function / homologyType 1 encapsulin shell protein / Encapsulating protein for peroxidase / : / encapsulin nanocompartment / iron ion transport / intracellular iron ion homeostasis / Type 1 encapsulin shell protein
Function and homology information
Biological speciesBacillus thermotolerans (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsAndreas, M.P. / Siddiquee, R. / Giessen, T.W. / Lau, Y.H.
Funding support Australia, United States, 3items
OrganizationGrant numberCountry
Australian Research Council (ARC)DP230101045 Australia
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM133325 United States
National Science Foundation (NSF, United States)2342136 United States
CitationJournal: bioRxiv / Year: 2026
Title: Directed evolution of multimeric proteins is enabled by dual-compensatory gene duplication.
Authors: Rezwan Siddiquee / Felicia Lie / Taylor N Szyszka / Alex Loustau / Michael P Andreas / Tobias W Giessen / Yu Heng Lau /
Abstract: Gene duplication has played a critical role in the evolutionary history of proteins, enabling complex multimers to emerge from simpler precursors. Yet in protein engineering, current methods for ...Gene duplication has played a critical role in the evolutionary history of proteins, enabling complex multimers to emerge from simpler precursors. Yet in protein engineering, current methods for directed evolution do not exploit gene duplication, hampering access to the vast array of diverse variants that are only enriched in the presence of a wild-type copy. We establish a directed evolution strategy for multimeric proteins that harnesses gene duplication to compensate for metabolic burden and self-assembly fitness, allowing previously inaccessible variants to be enriched. Starting from a homomeric 240-mer capsid, gene duplication enables selection of both extreme homomeric variants and obligate heteromers. This strategy significantly expands engineering access to diverse high-performing variants, while also supporting a plausible model for evolutionary diversification of higher-order multimers in nature.
History
DepositionDec 31, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 1, 2026Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Type 1 encapsulin shell protein
B: Type 1 encapsulin shell protein
C: Type 1 encapsulin shell protein
D: Type 1 encapsulin shell protein


Theoretical massNumber of molelcules
Total (without water)122,1704
Polymers122,1704
Non-polymers00
Water00
1
A: Type 1 encapsulin shell protein
B: Type 1 encapsulin shell protein
C: Type 1 encapsulin shell protein
D: Type 1 encapsulin shell protein
x 60


Theoretical massNumber of molelcules
Total (without water)7,330,195240
Polymers7,330,195240
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
B: Type 1 encapsulin shell protein
C: Type 1 encapsulin shell protein
D: Type 1 encapsulin shell protein
x 5


  • icosahedral pentamer
  • 611 kDa, 20 polymers
Theoretical massNumber of molelcules
Total (without water)610,85020
Polymers610,85020
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: Type 1 encapsulin shell protein
B: Type 1 encapsulin shell protein
C: Type 1 encapsulin shell protein
D: Type 1 encapsulin shell protein
x 6


  • icosahedral 23 hexamer
  • 733 kDa, 24 polymers
Theoretical massNumber of molelcules
Total (without water)733,01924
Polymers733,01924
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 / IMEF encapsulin


Mass: 30542.479 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus thermotolerans (bacteria) / Gene: enc, QY95_01592 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0F5HPP7
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

ComponentName: Quasibacillus thermotolerans T=4 encapsulin pore mutant variant Letter11
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Bacillus thermotolerans (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8 / Details: 200 mM NaCl, 25 mM Tris pH 8.0, 1 mM TCEP
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaCl1
225 mMTrisC4H11NO31
31 mMTCEPC9H15O6P1
SpecimenConc.: 4.5 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 R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1200 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 51.5 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1565

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Processing

EM software
IDNameVersionCategoryDetails (eV)
1cryoSPARC4.7.1particle selectionTemplate Picker
2SerialEMimage acquisition
4cryoSPARC4.7.1CTF correctionCTF estimation was performed using patch CTF estimation in cryoSPARC Live.
7UCSF ChimeraX1.8model fitting
9Coot0.9.8.1model refinement
10PHENIX1.20.1-4487-000model refinement
11cryoSPARC4.7.1initial Euler assignmentAb-initio reconstruction
12cryoSPARC4.7.1final Euler assignmentHomogeneous refinement
14cryoSPARC4.7.13D reconstructionHomogeneous Refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 68033
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 44759
Details: The final map was generated using homogeneous refinement against the ab-initio map with I symmetry imposed, per-particle defocus optimization, per-group CTF parameterization, spherical ...Details: The final map was generated using homogeneous refinement against the ab-initio map with I symmetry imposed, per-particle defocus optimization, per-group CTF parameterization, spherical aberration fitting enabled, tetrafoil fitting enabled, anisotropic magnification fitting enabled, and Ewald sphere correcting enabled with a negative curvature sign.
Symmetry type: POINT
Atomic model buildingB value: 79.3 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation coefficient
Details: A starting model of a single protomer was generated using AlphaFold 3, and individual protomers were fit into the volume of an asymmetric unit using UCSF ChimeraX v 1.8. The model containing ...Details: A starting model of a single protomer was generated using AlphaFold 3, and individual protomers were fit into the volume of an asymmetric unit using UCSF ChimeraX v 1.8. The model containing a single asymmetric unit consisting of four protomers was then manually refined using Coot v 0.9.8.1, followed by real-space refinement in PHENIX v 1.20.1-4487-000. Non-crystallographic symmetry (NCS) operators were then applied to generate a complete NCS-expanded shell, which was refined against the map using PHENIX real-space refinement.
Atomic model buildingSource name: AlphaFold / Type: in silico model

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