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- EMDB-41322: Myxococcus xanthus EncA protein shell with compartmentalized SNAP... -

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Basic information

Entry
Database: EMDB / ID: EMD-41322
TitleMyxococcus xanthus EncA protein shell with compartmentalized SNAP-tag cargo protein
Map dataMyxococcus xanthus EncA protein shell with compartmentalized SNAP-tag cargo protein
Sample
  • Complex: Myxococcus xanthus EncA protein shell with compartmentalized SNAP-tag cargo protein
    • Complex: Myxococcus xanthus EncA encapsulin shell protein
      • Protein or peptide: Type 1 encapsulin shell protein EncA
    • Complex: SNAP-Tag/EncC targeting peptide
      • Protein or peptide: Methylated-DNA--protein-cysteine methyltransferase
KeywordsEncapsulin / nanocompartment / VIRUS LIKE PARTICLE
Function / homology
Function and homology information


methylated-DNA-[protein]-cysteine S-methyltransferase / methylated-DNA-[protein]-cysteine S-methyltransferase activity / encapsulin nanocompartment / DNA modification / iron ion transport / methylation / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.53 Å
AuthorsAndreas MP / Kwon S / Giessen TW
Funding support United States, 1 items
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, 2023-
Header (metadata) releaseSep 13, 2023-
Map releaseSep 13, 2023-
UpdateSep 20, 2023-
Current statusSep 20, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41322.png

Downloads & links

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Map

FileDownload / File: emd_41322.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMyxococcus xanthus EncA protein shell with compartmentalized SNAP-tag cargo protein
Voxel sizeX=Y=Z: 1.10345 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.2792054 - 1.9937683
Average (Standard dev.)0.0140174655 (±0.11786885)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 485.51996 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map 1

Fileemd_41322_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_41322_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Myxococcus xanthus EncA protein shell with compartmentalized SNAP...

EntireName: Myxococcus xanthus EncA protein shell with compartmentalized SNAP-tag cargo protein
Components
  • Complex: Myxococcus xanthus EncA protein shell with compartmentalized SNAP-tag cargo protein
    • Complex: Myxococcus xanthus EncA encapsulin shell protein
      • Protein or peptide: Type 1 encapsulin shell protein EncA
    • Complex: SNAP-Tag/EncC targeting peptide
      • Protein or peptide: Methylated-DNA--protein-cysteine methyltransferase

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Supramolecule #1: Myxococcus xanthus EncA protein shell with compartmentalized SNAP...

SupramoleculeName: Myxococcus xanthus EncA protein shell with compartmentalized SNAP-tag cargo protein
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Myxococcus xanthus DK 1622 (bacteria)

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Supramolecule #2: Myxococcus xanthus EncA encapsulin shell protein

SupramoleculeName: Myxococcus xanthus EncA encapsulin shell protein / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: Recombinantly expressed Myxococcus xanthus EncA encapsulin shell with T=3 icosahedral symmetry
Source (natural)Organism: Myxococcus xanthus DK 1622 (bacteria)

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Supramolecule #3: SNAP-Tag/EncC targeting peptide

SupramoleculeName: SNAP-Tag/EncC targeting peptide / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Details: Internalized SNAP-tag/EncC targeting peptide cargo protein
Source (natural)Organism: Myxococcus xanthus DK 1622 (bacteria)

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Macromolecule #1: Type 1 encapsulin shell protein EncA

MacromoleculeName: Type 1 encapsulin shell protein EncA / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Myxococcus xanthus DK 1622 (bacteria)
Molecular weightTheoretical: 31.691977 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MPDFLGHAEN PLREEEWARL NETVIQVARR SLVGRRILDI YGPLGAGVQT VPYDEFQGVS PGAVDIVGEQ ETAMVFTDAR KFKTIPIIY KDFLLHWRDI EAARTHNMPL DVSAAAGAAA LCAQQEDELI FYGDARLGYE GLMTANGRLT VPLGDWTSPG G GFQAIVEA ...String:
MPDFLGHAEN PLREEEWARL NETVIQVARR SLVGRRILDI YGPLGAGVQT VPYDEFQGVS PGAVDIVGEQ ETAMVFTDAR KFKTIPIIY KDFLLHWRDI EAARTHNMPL DVSAAAGAAA LCAQQEDELI FYGDARLGYE GLMTANGRLT VPLGDWTSPG G GFQAIVEA TRKLNEQGHF GPYAVVLSPR LYSQLHRIYE KTGVLEIETI RQLASDGVYQ SNRLRGESGV VVSTGRENMD LA VSMDMVA AYLGASRMNH PFRVLEALLL RIKHPDAICT LEGAGATERR

UniProtKB: Type 1 encapsulin shell protein EncA

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Macromolecule #2: Methylated-DNA--protein-cysteine methyltransferase

MacromoleculeName: Methylated-DNA--protein-cysteine methyltransferase / type: protein_or_peptide / ID: 2
Details: Amino acids 1-183 are unresolved SNAP-TAG. Amino acids 184-191 are unresolved linker. Amino acids 192-203 are EncC targeting peptide.
Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 21.4826 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGPGSDKDCE MKRTTLDSPL GKLELSGCEQ GLHEIIFLGK GTSAADAVEV PAPAAVLGGP EPLMQATAWL NAYFHQPEAI EEFPVPALH HPVFQQESFT RQVLWKLLKV VKFGEVISYS HLAALAGNPA ATAAVKTALS GNPVPILIPC HRVVQGDLDV G GYEGGLAV ...String:
MGPGSDKDCE MKRTTLDSPL GKLELSGCEQ GLHEIIFLGK GTSAADAVEV PAPAAVLGGP EPLMQATAWL NAYFHQPEAI EEFPVPALH HPVFQQESFT RQVLWKLLKV VKFGEVISYS HLAALAGNPA ATAAVKTALS GNPVPILIPC HRVVQGDLDV G GYEGGLAV KEWLLAHEGH RLGKRGGGSG GGSPEKRLTV GSLRR

UniProtKB: Methylated-DNA--protein-cysteine methyltransferase

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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

Concentration3.4 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
20.0 mMC4H12NO32-amino-2-hydroxymethyl-propane-1,3-diol

Details: 150 mM NaCl, 20 mM Tris pH 7.5
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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 4092 pixel / Digitization - Dimensions - Height: 5760 pixel / Number grids imaged: 1 / Number real images: 2610 / Average exposure time: 3.2 sec. / Average electron dose: 49.26 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: Ab-initio reconstruction
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 4.2.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 4.2.1)
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 2.53 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Software - details: Non-uniform refinement / Number images used: 50680
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7s2t


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsThe 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.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 89.9 / Target criteria: Cross-correlation coefficient
Output model

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

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