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- PDB-8tk7: Myxococcus xanthus EncA protein shell with compartmentalized SNAP... -

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Basic information

Entry
Database: PDB / ID: 8tk7
TitleMyxococcus xanthus EncA protein shell with compartmentalized SNAP-tag cargo protein
Components
  • Methylated-DNA--protein-cysteine methyltransferase
  • Type 1 encapsulin shell protein EncA
KeywordsVIRUS LIKE PARTICLE / Encapsulin / nanocompartment
Function / homology
Function and homology information


methylated-DNA-[protein]-cysteine S-methyltransferase / methylated-DNA-[protein]-cysteine S-methyltransferase activity / encapsulin nanocompartment / DNA modification / methylation / iron ion transport / intracellular iron ion homeostasis / DNA repair / DNA binding / metal ion binding / nucleus
Similarity search - Function
Methylguanine DNA methyltransferase, ribonuclease-like domain / 6-O-methylguanine DNA methyltransferase, ribonuclease-like domain / Methylated DNA-protein cysteine methyltransferase domain superfamily / Methylated-DNA-[protein]-cysteine S-methyltransferase, active site / Methylated-DNA--protein-cysteine methyltransferase active site. / Methylated-DNA-[protein]-cysteine S-methyltransferase, DNA binding / Methylated DNA-protein cysteine methyltransferase, DNA binding domain / 6-O-methylguanine DNA methyltransferase, DNA binding domain / Type 1 encapsulin shell protein / Encapsulating protein for peroxidase / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
Methylated-DNA--protein-cysteine methyltransferase / Type 1 encapsulin shell protein EncA
Similarity search - Component
Biological speciesMyxococcus xanthus DK 1622 (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.53 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)R35GM133325 United States
CitationJournal: J Struct Biol / Year: 2023
Title: Structure and heterogeneity of a highly cargo-loaded encapsulin shell.
Authors: Seokmu Kwon / Michael P Andreas / Tobias W Giessen /
Abstract: Encapsulins are self-assembling protein nanocompartments able to selectively encapsulate dedicated cargo enzymes. Encapsulins are widespread across bacterial and archaeal phyla and are involved in ...Encapsulins are self-assembling protein nanocompartments able to selectively encapsulate dedicated cargo enzymes. Encapsulins are widespread across bacterial and archaeal phyla and are involved in oxidative stress resistance, iron storage, and sulfur metabolism. Encapsulin shells exhibit icosahedral geometry and consist of 60, 180, or 240 identical protein subunits. Cargo encapsulation is mediated by the specific interaction of targeting peptides or domains, found in all cargo proteins, with the interior surface of the encapsulin shell during shell self-assembly. Here, we report the 2.53 Å cryo-EM structure of a heterologously produced and highly cargo-loaded T3 encapsulin shell from Myxococcus xanthus and explore the systems' structural heterogeneity. We find that exceedingly high cargo loading results in the formation of substantial amounts of distorted and aberrant shells, likely caused by a combination of unfavorable steric clashes of cargo proteins and shell conformational changes. Based on our cryo-EM structure, we determine and analyze the targeting peptide-shell binding mode. We find that both ionic and hydrophobic interactions mediate targeting peptide binding. Our results will guide future attempts at rationally engineering encapsulins for biomedical and biotechnological applications.
History
DepositionJul 25, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 13, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 20, 2023Group: Database references / Refinement description / Category: citation / em_3d_fitting_list
Item: _citation.journal_volume / _em_3d_fitting_list.initial_refinement_model_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Type 1 encapsulin shell protein EncA
A: Type 1 encapsulin shell protein EncA
C: Type 1 encapsulin shell protein EncA
E: Methylated-DNA--protein-cysteine methyltransferase
D: Methylated-DNA--protein-cysteine methyltransferase
F: Methylated-DNA--protein-cysteine methyltransferase


Theoretical massNumber of molelcules
Total (without water)159,5246
Polymers159,5246
Non-polymers00
Water0
1
B: Type 1 encapsulin shell protein EncA
A: Type 1 encapsulin shell protein EncA
C: Type 1 encapsulin shell protein EncA
E: Methylated-DNA--protein-cysteine methyltransferase
D: Methylated-DNA--protein-cysteine methyltransferase
F: Methylated-DNA--protein-cysteine methyltransferase
x 60


Theoretical massNumber of molelcules
Total (without water)9,571,424360
Polymers9,571,424360
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
B: Type 1 encapsulin shell protein EncA
A: Type 1 encapsulin shell protein EncA
C: Type 1 encapsulin shell protein EncA
E: Methylated-DNA--protein-cysteine methyltransferase
D: Methylated-DNA--protein-cysteine methyltransferase
F: Methylated-DNA--protein-cysteine methyltransferase
x 5


  • icosahedral pentamer
  • 798 kDa, 30 polymers
Theoretical massNumber of molelcules
Total (without water)797,61930
Polymers797,61930
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
B: Type 1 encapsulin shell protein EncA
A: Type 1 encapsulin shell protein EncA
C: Type 1 encapsulin shell protein EncA
E: Methylated-DNA--protein-cysteine methyltransferase
D: Methylated-DNA--protein-cysteine methyltransferase
F: Methylated-DNA--protein-cysteine methyltransferase
x 6


  • icosahedral 23 hexamer
  • 957 kDa, 36 polymers
Theoretical massNumber of molelcules
Total (without water)957,14236
Polymers957,14236
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


Mass: 31691.977 Da / Num. of mol.: 3
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
#2: Protein Methylated-DNA--protein-cysteine methyltransferase


Mass: 21482.600 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: Amino acids 1-183 are unresolved SNAP-TAG. Amino acids 184-191 are unresolved linker. Amino acids 192-203 are EncC targeting peptide.
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: E5BBQ0

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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-IDSourceDetails
1Myxococcus xanthus EncA protein shell with compartmentalized SNAP-tag cargo proteinCOMPLEXall0RECOMBINANT
2Myxococcus xanthus EncA encapsulin shell proteinCOMPLEX#11RECOMBINANTRecombinantly expressed Myxococcus xanthus EncA encapsulin shell with T=3 icosahedral symmetry
3SNAP-Tag/EncC targeting peptideCOMPLEX#21RECOMBINANTInternalized SNAP-tag/EncC targeting peptide cargo protein
Molecular weight
IDEntity assembly-IDExperimental value
11NO
22
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 chlorideNaClSodium chloride1
220 mM2-amino-2-hydroxymethyl-propane-1,3-diolC4H12NO31
SpecimenConc.: 3.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid 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: 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: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.2 sec. / Electron dose: 49.26 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2610
Image scansWidth: 4092 / Height: 5760

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.2.1particle selectionTemplate picker
2SerialEMimage acquisition
4cryoSPARC4.2.1CTF correctionPatch CTF
7UCSF ChimeraX1.15model fitting
9cryoSPARC4.2.1initial Euler assignment
10cryoSPARC4.2.1final Euler assignment
12cryoSPARC4.2.13D reconstructionNon-uniform refinement
13Coot0.9.8.1model refinement
14PHENIX1.20.1-4487model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 2.53 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50680 / Symmetry type: POINT
Atomic model buildingB value: 89.9 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: The model was initially fit into the map using UCSF ChimeraX. It was then manually refined using Coot, followed by real-space refinement against the map using Phenix.
Atomic model buildingPDB-ID: 7S2T

7s2t


Accession code: 7S2T / Source name: PDB / Type: experimental model

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