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- PDB-8v4q: Myxococcus xanthus EncA 3xHis pore mutant with tetrahedral symmetry -

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Basic information

Entry
Database: PDB / ID: 8v4q
TitleMyxococcus xanthus EncA 3xHis pore mutant with tetrahedral symmetry
ComponentsType 1 encapsulin shell protein EncA
KeywordsVIRUS LIKE PARTICLE / Encapsulin
Function / homologyType 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: 2.71 Å
AuthorsSzyszka, T.N. / Andreas, M.P. / Lie, F. / Miller, L.M. / Adamson, L.S.R. / Fatehi, F. / Twarock, R. / Draper, B.E. / Jarrold, M.F. / Giessen, T.W. / Lau, Y.H.
Funding support Australia, United States, 4items
OrganizationGrant numberCountry
Australian Research Council (ARC)DE19010062 Australia
Australian Research Council (ARC)DP230101045 Australia
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM133325 United States
Other privateWestpac Scholars Trust WRF2020 Australia
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Point mutation in a virus-like capsid drives symmetry reduction to form tetrahedral cages.
Authors: Taylor N Szyszka / Michael P Andreas / Felicia Lie / Lohra M Miller / Lachlan S R Adamson / Farzad Fatehi / Reidun Twarock / Benjamin E Draper / Martin F Jarrold / Tobias W Giessen / Yu Heng Lau /
Abstract: Protein capsids are a widespread form of compartmentalization in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximizes the internal volume ...Protein capsids are a widespread form of compartmentalization in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximizes the internal volume that can be enclosed within. Despite the strong preference for icosahedral symmetry, we show that simple point mutations in a virus-like capsid can drive the assembly of unique symmetry-reduced structures. Starting with the encapsulin from , a 180-mer bacterial capsid that adopts the well-studied viral HK97 fold, we use mass photometry and native charge detection mass spectrometry to identify a triple histidine point mutant that forms smaller dimorphic assemblies. Using cryoelectron microscopy, we determine the structures of a precedented 60-mer icosahedral assembly and an unexpected 36-mer tetrahedron that features significant geometric rearrangements around a new interaction surface between capsid protomers. We subsequently find that the tetrahedral assembly can be generated by triple-point mutation to various amino acids and that even a single histidine point mutation is sufficient to form tetrahedra. These findings represent a unique example of tetrahedral geometry when surveying all characterized encapsulins, HK97-like capsids, or indeed any virus-derived capsids reported in the Protein Data Bank, revealing the surprising plasticity of capsid self-assembly that can be accessed through minimal changes in the protein sequence.
History
DepositionNov 29, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 22, 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


Theoretical massNumber of molelcules
Total (without water)95,4573
Polymers95,4573
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, gel filtration, light scattering, mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Type 1 encapsulin shell protein EncA


Mass: 31819.082 Da / Num. of mol.: 3 / Mutation: K199H, T200H, G201H
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Myxococcus xanthus DK 1622 (bacteria) / Strain: DK1622 / Gene: encA / 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 3xHis pore mutant with tetrahedral symmetry
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.14 MDa / Experimental value: YES
Source (natural)Organism: Myxococcus xanthus DK 1622 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8 / Details: 50 mM Tris pH 8.0, 200 mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMtrisC4H11NO31
2200 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 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 / Details: Blot force: 5 Blot time: 2 seconds

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.0972 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3765
Image scansWidth: 4092 / Height: 5760

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.2.1particle selection
2SerialEMimage acquisition
4cryoSPARC4.2.1CTF correction
7UCSF ChimeraX1.25model fitting
9cryoSPARC4.2.1initial Euler assignment
10cryoSPARC4.2.1final Euler assignment
12cryoSPARC4.2.13D reconstructionHomogenous refinement
13Coot0.9.8.1model refinement
14PHENIX1.20.1-4487model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 495482
3D reconstructionResolution: 2.71 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2799588
Details: Local refinement of the asymmetric unit containing three copies of the protomer from a starting map with tetrahedral symmetry. The particles used for refinement were generated by symmetry ...Details: Local refinement of the asymmetric unit containing three copies of the protomer from a starting map with tetrahedral symmetry. The particles used for refinement were generated by symmetry expansion using tetrahedral symmetry from 233,299 particles.
Symmetry type: POINT
Atomic model buildingB value: 118.3 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: The starting model was generated using ESMfold then fit into the map using ChimeraX. The placed coordinates were then manually refined using Coot, followed by real space refinement in Phenix.
Atomic model buildingDetails: ESMfold was used to make starting model / Source name: Other / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0026118
ELECTRON MICROSCOPYf_angle_d0.4428294
ELECTRON MICROSCOPYf_dihedral_angle_d3.777846
ELECTRON MICROSCOPYf_chiral_restr0.043917
ELECTRON MICROSCOPYf_plane_restr0.0041087

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