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- PDB-8dnl: Acidipropionibacterium acidipropionici encapsulin in an open stat... -

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Basic information

Entry
Database: PDB / ID: 8dnl
TitleAcidipropionibacterium acidipropionici encapsulin in an open state at pH 7.5
Components29 kDa antigen cfp29
KeywordsVIRUS LIKE PARTICLE / Encapsulin
Function / homologyType 1 encapsulin shell protein / Encapsulating protein for peroxidase / encapsulin nanocompartment / 29 kDa antigen cfp29
Function and homology information
Biological speciesAcidipropionibacterium acidipropionici ATCC 4875 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.32 Å
AuthorsJones, J.A. / Andreas, 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: Biomacromolecules / Year: 2023
Title: Exploring the Extreme Acid Tolerance of a Dynamic Protein Nanocage.
Authors: Jesse A Jones / Michael P Andreas / Tobias W Giessen /
Abstract: Encapsulins are microbial protein nanocages capable of efficient self-assembly and cargo enzyme encapsulation. Due to their favorable properties, including high thermostability, protease resistance, ...Encapsulins are microbial protein nanocages capable of efficient self-assembly and cargo enzyme encapsulation. Due to their favorable properties, including high thermostability, protease resistance, and robust heterologous expression, encapsulins have become popular bioengineering tools for applications in medicine, catalysis, and nanotechnology. Resistance against physicochemical extremes like high temperature and low pH is a highly desirable feature for many biotechnological applications. However, no systematic search for acid-stable encapsulins has been carried out, while the influence of pH on encapsulin shells has so far not been thoroughly explored. Here, we report on a newly identified encapsulin nanocage from the acid-tolerant bacterium . Using transmission electron microscopy, dynamic light scattering, and proteolytic assays, we demonstrate its extreme acid tolerance and resilience against proteases. We structurally characterize the novel nanocage using cryo-electron microscopy, revealing a dynamic five-fold pore that displays distinct "closed" and "open" states at neutral pH but only a singular "closed" state under strongly acidic conditions. Further, the "open" state exhibits the largest pore in an encapsulin shell reported to date. Non-native protein encapsulation capabilities are demonstrated, and the influence of external pH on internalized cargo is explored. Our results expand the biotechnological application range of encapsulin nanocages toward potential uses under strongly acidic conditions and highlight pH-responsive encapsulin pore dynamics.
History
DepositionJul 11, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 22, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: 29 kDa antigen cfp29


Theoretical massNumber of molelcules
Total (without water)28,5231
Polymers28,5231
Non-polymers00
Water0
1
A: 29 kDa antigen cfp29
x 60


Theoretical massNumber of molelcules
Total (without water)1,711,36460
Polymers1,711,36460
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
A: 29 kDa antigen cfp29
x 5


  • icosahedral pentamer
  • 143 kDa, 5 polymers
Theoretical massNumber of molelcules
Total (without water)142,6145
Polymers142,6145
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: 29 kDa antigen cfp29
x 6


  • icosahedral 23 hexamer
  • 171 kDa, 6 polymers
Theoretical massNumber of molelcules
Total (without water)171,1366
Polymers171,1366
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 29 kDa antigen cfp29 / Encapsulin


Mass: 28522.725 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acidipropionibacterium acidipropionici ATCC 4875 (bacteria)
Strain: ATCC 4875 / DSM 20272 / JCM 6432 / NBRC 12425 / NCIMB 8070 / 4
Gene: PACID_10050 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: K7RV67

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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: Acidipropionibacterium acidipropionici encapsulin in an open state at pH 7.5
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.71 MDa / Experimental value: YES
Source (natural)Organism: Acidipropionibacterium acidipropionici ATCC 4875 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaClSodium chloride1
220 mMTrisC4H11NO31
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: The grid was glow discharged for 60 seconds at 5 mA.
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: Additional settings were : Blot force- 20, blot time - 4 seconds, drain time - 0 seconds, wait time - 0 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 FIELDBright-field microscopy / Nominal magnification: 45000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 4 sec. / Electron dose: 41 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 975
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 20

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.1particle selection
2Leginonimage acquisition
4cryoSPARC3.3.1CTF correction
7PHENIXmodel fitting
8UCSF Chimeramodel fitting
10cryoSPARC3.3.1initial Euler assignment
11cryoSPARC3.3.1final Euler assignment
13cryoSPARC3.3.13D reconstruction
14Cootmodel refinement
15PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 13581 / Symmetry type: POINT
Atomic model buildingB value: 77.81 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient
Details: A starting model was created using RosettaFold. The starting model was placed in the map manually using UCSF Chimera. The model was then further refined using Coot and Phenix Real Space Refine.

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