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- PDB-9bhv: Streptomyces griseus Family 2B encapsulin shell with 2-methylisob... -

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Basic information

Entry
Database: PDB / ID: 9bhv
TitleStreptomyces griseus Family 2B encapsulin shell with 2-methylisoborneol synthase cargo
ComponentsNucleotide-binding protein
KeywordsVIRUS LIKE PARTICLE / Encapsulin / nanocompartment
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.02 Å
AuthorsAndreas, M.P. / 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: 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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 27, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Nucleotide-binding protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)52,0542
Polymers52,0141
Non-polymers401
Water00
1
A: Nucleotide-binding protein
hetero molecules
x 60


Theoretical massNumber of molelcules
Total (without water)3,123,253120
Polymers3,120,84860
Non-polymers2,40560
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
A: Nucleotide-binding protein
hetero molecules
x 5


  • icosahedral pentamer
  • 260 kDa, 5 polymers
Theoretical massNumber of molelcules
Total (without water)260,27110
Polymers260,0715
Non-polymers2005
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: Nucleotide-binding protein
hetero molecules
x 6


  • icosahedral 23 hexamer
  • 312 kDa, 6 polymers
Theoretical massNumber of molelcules
Total (without water)312,32512
Polymers312,0856
Non-polymers2406
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 Nucleotide-binding protein / Sporulation protein


Mass: 52014.137 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Residues 1-39, 147-152, and 216-245 are not resolved in the map
Source: (gene. exp.) Streptomyces griseus subsp. griseus (bacteria)
Gene: mmpI_1, NCTC13033_02226 / Production host: Streptomyces coelicolor A3(2) (bacteria) / References: UniProt: Q54255
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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-IDSource
1Streptomyces griseus Family 2B encapsulin shell with 2-methylisoborneol synthase cargoCOMPLEX#10RECOMBINANT
22-Methylisoborneol synthase cargo proteinCOMPLEX1RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
22
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Streptomyces griseus subsp. griseus (bacteria)67263
32Streptomyces griseus subsp. griseus (bacteria)67263
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
21Streptomyces coelicolor A3(2) (bacteria)100226John Innes Centre M145
32Streptomyces coelicolor A3(2) (bacteria)100226John Innes Centre M145
Buffer solutionpH: 7.5 / Details: 150 mM NaCl, 20 mM Tris pH 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMtrisC4H11NO31
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 60 seconds at 5 mA under vacuum / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K / Details: Blot force: 20 Blot time: 4 seconds

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 45000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm
Image recordingAverage exposure time: 8 sec. / Electron dose: 42.94 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1
Image scansWidth: 3710 / Height: 3838 / Movie frames/image: 40

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.4.0+231114particle selectionTemplate Picker
2Leginonimage acquisition
4cryoSPARC4.4.0+231114CTF correctionPatch CTF Estimation
7UCSF ChimeraX1.5model fitting
9cryoSPARC4.4.0+231114initial Euler assignment
10cryoSPARC4.4.0+231114final Euler assignment
12cryoSPARC4.4.0+2311143D reconstructionHomogeneous Refinement
13Coot8.9.1model refinement
14PHENIX1.20.1-4487model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 54206
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51173
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.
Symmetry type: POINT
Atomic model buildingB value: 115.8 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation coefficient
Details: An 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 ...Details: An 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.
Atomic model buildingAccession code: Q54255 / Source name: AlphaFold / Type: in silico model

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