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- EMDB-44554: Streptomyces griseus Family 2B encapsulin shell with 2-methylisob... -

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Basic information

Entry
Database: EMDB / ID: EMD-44554
TitleStreptomyces griseus Family 2B encapsulin shell with 2-methylisoborneol synthase cargo
Map dataStreptomyces griseus Family 2B encapsulin shell with 2-methylisoborneol synthase cargo
Sample
  • Complex: Streptomyces griseus Family 2B encapsulin shell with 2-methylisoborneol synthase cargo
    • Complex: 2-Methylisoborneol synthase cargo protein
    • Protein or peptide: Nucleotide-binding protein
  • Ligand: CALCIUM ION
KeywordsEncapsulin / nanocompartment / VIRUS LIKE PARTICLE
Function / homology
Function and homology information


DNA-binding transcription factor activity / cytosol
Similarity search - Function
: / Type 2A encapsulin shell protein SrpI-like / Type 2A encapsulin shell protein SrpI-like / : / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / RmlC-like jelly roll fold
Similarity search - Domain/homology
Biological speciesStreptomyces griseus subsp. griseus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.02 Å
AuthorsAndreas MP / 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: Nat Commun / Year: 2024
Title: The biosynthesis of the odorant 2-methylisoborneol is compartmentalized inside a protein shell.
Authors: Michael P Andreas / Tobias W Giessen /
Abstract: Terpenoids are the largest class of natural products, found across all domains of life. One of the most abundant bacterial terpenoids is the volatile odorant 2-methylisoborneol (2-MIB), partially ...Terpenoids are the largest class of natural products, found across all domains of life. One of the most abundant bacterial terpenoids is the volatile odorant 2-methylisoborneol (2-MIB), partially responsible for the earthy smell of soil and musty taste of contaminated water. Many bacterial 2-MIB biosynthetic gene clusters were thought to encode a conserved transcription factor, named EshA in the model soil bacterium Streptomyces griseus. Here, we revise the function of EshA, now referred to as Sg Enc, and show that it is a Family 2B encapsulin shell protein. Using cryo-electron microscopy, we find that Sg Enc forms an icosahedral protein shell and encapsulates 2-methylisoborneol synthase (2-MIBS) as a cargo protein. Sg Enc contains a cyclic adenosine monophosphate (cAMP) binding domain (CBD)-fold insertion and a unique metal-binding domain, both displayed on the shell exterior. We show that Sg Enc CBDs do not bind cAMP. We find that 2-MIBS cargo loading is mediated by an N-terminal disordered cargo-loading domain and that 2-MIBS activity and Sg Enc shell structure are not modulated by cAMP. Our work redefines the function of EshA and establishes Family 2B encapsulins as cargo-loaded protein nanocompartments involved in secondary metabolite biosynthesis.
History
DepositionApr 22, 2024-
Header (metadata) releaseNov 27, 2024-
Map releaseNov 27, 2024-
UpdateNov 27, 2024-
Current statusNov 27, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44554.png

Downloads & links

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Map

FileDownload / File: emd_44554.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStreptomyces griseus Family 2B encapsulin shell with 2-methylisoborneol synthase cargo
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.91 Å/pix.
x 440 pix.
= 400.4 Å		0.91 Å/pix.
x 440 pix.
= 400.4 Å		0.91 Å/pix.
x 440 pix.
= 400.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.91 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.4123714 - 0.75568706
Average (Standard dev.)0.003309398 (±0.03685446)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 400.40002 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: This map is a homogenous reconstruction of a...

Fileemd_44554_additional_1.map
AnnotationThis map is a homogenous reconstruction of a 3D class from 3D classification using a focus mask that includes 2 cyclic-nucleotide binding domains with C1 symmetry.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_44554_half_map_1.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B

Fileemd_44554_half_map_2.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Streptomyces griseus Family 2B encapsulin shell with 2-methylisob...

EntireName: Streptomyces griseus Family 2B encapsulin shell with 2-methylisoborneol synthase cargo
Components
  • Complex: Streptomyces griseus Family 2B encapsulin shell with 2-methylisoborneol synthase cargo
    • Complex: 2-Methylisoborneol synthase cargo protein
    • Protein or peptide: Nucleotide-binding protein
  • Ligand: CALCIUM ION

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Supramolecule #1: Streptomyces griseus Family 2B encapsulin shell with 2-methylisob...

SupramoleculeName: Streptomyces griseus Family 2B encapsulin shell with 2-methylisoborneol synthase cargo
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Streptomyces griseus subsp. griseus (bacteria)

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Supramolecule #2: 2-Methylisoborneol synthase cargo protein

SupramoleculeName: 2-Methylisoborneol synthase cargo protein / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Streptomyces griseus subsp. griseus (bacteria)

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Macromolecule #1: Nucleotide-binding protein

MacromoleculeName: Nucleotide-binding protein / type: protein_or_peptide / ID: 1
Details: Residues 1-39, 147-152, and 216-245 are not resolved in the map
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Streptomyces griseus subsp. griseus (bacteria)
Molecular weightTheoretical: 52.014137 KDa
Recombinant expressionOrganism: Streptomyces coelicolor A3(2) (bacteria)
SequenceString: MTVDSTSEAR LEVPRQSSLG TAAARNLAST TKSAPQMQEI TSRWLLRMLP WVETKGGAYR VNRRLTFTVG DGRVEFVQDG STVRVIPQE LGELALLRDF DDAEVLSAIA DRCVQRDFRA GETLVERGTA ADELHLIAHG RIGQASAGSY GDEVTLDVLA D GDRFGEHA ...String:
MTVDSTSEAR LEVPRQSSLG TAAARNLAST TKSAPQMQEI TSRWLLRMLP WVETKGGAYR VNRRLTFTVG DGRVEFVQDG STVRVIPQE LGELALLRDF DDAEVLSAIA DRCVQRDFRA GETLVERGTA ADELHLIAHG RIGQASAGSY GDEVTLDVLA D GDRFGEHA LLDEDARWSQ TATAETSGTL LTLSRADFAA VVANSPALRS HLAAFTARSE QRQNHRGEAE IAMSAGHVGE HE LPGAFAD YELKPREYEL SVAQTILRVH TRVADLYNGP MNQTEEQLRL TIEALRERQE HELINNREFG LLHNADFKQR IQT HSGPPT PDDLDELLCR RRGTKFFLAH PRTIAAMGRE FNARGLYPDH TDLGGQQVPA WRGVPILPCG KIPITPERTS SILA LRTGE EDQGVIGLRQ TGLPDEYEPG LSVRFMNIDE KAIISYLVST YYSAAILVPD AVGVLENVQI ANWPR

UniProtKB: Sporulation protein

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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

Concentration3.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMC4H11NO3tris

Details: 150 mM NaCl, 20 mM Tris pH 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Details: 60 seconds at 5 mA under vacuum
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV / Details: Blot force: 20 Blot time: 4 seconds.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Number grids imaged: 1 / Average exposure time: 8.0 sec. / Average electron dose: 42.94 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 45000
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 54206
Startup modelType of model: INSILICO MODEL / In silico model: Ab-initio model
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.0+231114) / Software - details: Homogeneous Refinement
Details: Homogeneous refinement was performed against an ab-initio reconstruction map with I symmetry imposed, per particle defocus optimization, per-group CTF parameterization, and Ewald sphere ...Details: Homogeneous refinement was performed against an ab-initio reconstruction map with I symmetry imposed, per particle defocus optimization, per-group CTF parameterization, and Ewald sphere correction enabled with a positive curvature sign.
Number images used: 51173
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.0+231114)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.0+231114)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4255


Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
DetailsAn AlphaFold-generated model was fit into the map using ChimeraX v1.5. The model was then refined iteratively by alternating rounds of manual refinement in Coot v8.9.1 and real-space refinements using PHENIX v1.20.1-4487.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 115.8 / Target criteria: cross-correlation coefficient
Output model

PDB-9bhv:
Streptomyces griseus Family 2B encapsulin shell with 2-methylisoborneol synthase cargo

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