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- EMDB-74905: Quasibacillus thermotolerans T=3 encapsulin pore mutant variant L... -

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Basic information

Entry
Database: EMDB / ID: EMD-74905
TitleQuasibacillus thermotolerans T=3 encapsulin pore mutant variant Letter11
Map data
Sample
  • Complex: Quasibacillus thermotolerans T=3 encapsulin pore mutant variant Letter11
    • Protein or peptide: Type 1 encapsulin shell protein
Keywordsencapsulin / protein nanocompartment / VIRUS LIKE PARTICLE
Biological speciesBacillus thermotolerans (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.42 Å
AuthorsAndreas MP / Siddiquee R / Giessen TW / Lau YH
Funding support Australia, United States, 3 items
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, 2025-
Header (metadata) releaseJul 1, 2026-
Map releaseJul 1, 2026-
UpdateJul 1, 2026-
Current statusJul 1, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_74905.png

Downloads & links

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Map

FileDownload / File: emd_74905.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.19 Å/pix.
x 480 pix.
= 570.456 Å		1.19 Å/pix.
x 480 pix.
= 570.456 Å		1.19 Å/pix.
x 480 pix.
= 570.456 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.18845 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.51
Minimum - Maximum-2.0322876 - 3.6813648
Average (Standard dev.)0.008179757 (±0.17109717)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 570.456 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_74905_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

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Histogram (log scale)

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Half map: #2

Fileemd_74905_half_map_2.map
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Sample components

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Entire : Quasibacillus thermotolerans T=3 encapsulin pore mutant variant L...

EntireName: Quasibacillus thermotolerans T=3 encapsulin pore mutant variant Letter11
Components
  • Complex: Quasibacillus thermotolerans T=3 encapsulin pore mutant variant Letter11
    • Protein or peptide: Type 1 encapsulin shell protein

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Supramolecule #1: Quasibacillus thermotolerans T=3 encapsulin pore mutant variant L...

SupramoleculeName: Quasibacillus thermotolerans T=3 encapsulin pore mutant variant Letter11
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bacillus thermotolerans (bacteria)

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Macromolecule #1: Type 1 encapsulin shell protein

MacromoleculeName: Type 1 encapsulin shell protein / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Bacillus thermotolerans (bacteria)
Molecular weightTheoretical: 30.542479 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNKSQLYPDS PLTDQDFNQL DQTVIEAARR QLVGRRFIEL YGPLGRGMQS VFNDIFMESH EAKMDFQGSF DTEVESSRRV NYTIPMLYK DFVLYWRDLE QSKALDIPID FSVAANAARD VAFLEDQMIF HGSKEFDIPG LMNVKGRLTH LIGNWYESGN A FQDIVEAR ...String:
MNKSQLYPDS PLTDQDFNQL DQTVIEAARR QLVGRRFIEL YGPLGRGMQS VFNDIFMESH EAKMDFQGSF DTEVESSRRV NYTIPMLYK DFVLYWRDLE QSKALDIPID FSVAANAARD VAFLEDQMIF HGSKEFDIPG LMNVKGRLTH LIGNWYESGN A FQDIVEAR NKLLEMNHNG PYALVLSPEL YSLLGSGGSG LITAGVFQSP VLKGKSGVIV NTGRNNLDLA ISEDFETAYL GE EGMNHPF RVYETVVLRI KRPAAICTLI DPEE

UniProtKB: Type 1 encapsulin shell protein, Type 1 encapsulin shell protein

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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

Concentration4.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
200.0 mMNaClsodium chloride
25.0 mMC4H11NO3Tris
1.0 mMC9H15O6PTCEP

Details: 200 mM NaCl, 25 mM Tris pH 8.0, 1 mM TCEP
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: The grid was glow discharged at 5 mA for 60 seconds under vacuum.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 1565 / Average electron dose: 51.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 68033
CTF correctionSoftware - Name: cryoSPARC (ver. 4.7.1)
Software - details: CTF estimation was performed using patch CTF estimation in cryoSPARC Live.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
In silico model: Starting model was generated using Ab-initio reconstruction with I symmetry.
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 2.42 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7.1) / Software - details: Homogeneous Refinement
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.
Number images used: 18327
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.1) / Software - details: Ab-initio reconstruction
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.1) / Software - details: Homogeneous refinement
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
DetailsA 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 three 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.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 71.2 / Target criteria: cross-correlation coefficient
Output model

PDB-9zw4:
Quasibacillus thermotolerans T=3 encapsulin pore mutant variant Letter11

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