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- EMDB-27573: Acidipropionibacterium acidipropionici encapsulin in an open stat... -

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Basic information

Entry
Database: EMDB / ID: EMD-27573
TitleAcidipropionibacterium acidipropionici encapsulin in an open state at pH 7.5
Map dataAcidipropionibacterium acidipropionici encapsulin in an open state at pH 7.5
Sample
  • Complex: Acidipropionibacterium acidipropionici encapsulin in an open state at pH 7.5
    • Protein or peptide: 29 kDa antigen cfp29
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.32 Å
AuthorsJones JA / Andreas 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: 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, 2022-
Header (metadata) releaseMar 22, 2023-
Map releaseMar 22, 2023-
UpdateMar 22, 2023-
Current statusMar 22, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27573.png

Downloads & links

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Map

FileDownload / File: emd_27573.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAcidipropionibacterium acidipropionici encapsulin in an open state at pH 7.5
Voxel sizeX=Y=Z: 0.91 Å
Density
Contour LevelBy AUTHOR: 0.16
Minimum - Maximum-0.28650287 - 0.57489145
Average (Standard dev.)0.0034501394 (±0.03561157)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 349.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Acidipropionibacterium acidipropionici encapsulin in an open state at...

Fileemd_27573_half_map_1.map
AnnotationAcidipropionibacterium acidipropionici encapsulin in an open state at pH 7.5
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Acidipropionibacterium acidipropionici encapsulin in an open state at...

Fileemd_27573_half_map_2.map
AnnotationAcidipropionibacterium acidipropionici encapsulin in an open state at pH 7.5
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Acidipropionibacterium acidipropionici encapsulin in an open stat...

EntireName: Acidipropionibacterium acidipropionici encapsulin in an open state at pH 7.5
Components
  • Complex: Acidipropionibacterium acidipropionici encapsulin in an open state at pH 7.5
    • Protein or peptide: 29 kDa antigen cfp29

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Supramolecule #1: Acidipropionibacterium acidipropionici encapsulin in an open stat...

SupramoleculeName: Acidipropionibacterium acidipropionici encapsulin in an open state at pH 7.5
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Acidipropionibacterium acidipropionici ATCC 4875 (bacteria)
Molecular weightTheoretical: 1.71 MDa

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Macromolecule #1: 29 kDa antigen cfp29

MacromoleculeName: 29 kDa antigen cfp29 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Acidipropionibacterium acidipropionici ATCC 4875 (bacteria)
Strain: ATCC 4875 / DSM 20272 / JCM 6432 / NBRC 12425 / NCIMB 8070 / 4
Molecular weightTheoretical: 28.522725 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNNLHRELAP ISEAAWKQID DEARDTFSLR AAGRRVVDVP EPAGPTLGSV SLGHLETGSQ TDGVQTSVYR VQPLVQVRVP FTVSRADID DVERGAVDLT WDPVDDAVAK LVDTEDTAIL HGWEEAGITG LSEASVHQPV QMPAELEQID DAVSGACNVL R LADVEGPY ...String:
MNNLHRELAP ISEAAWKQID DEARDTFSLR AAGRRVVDVP EPAGPTLGSV SLGHLETGSQ TDGVQTSVYR VQPLVQVRVP FTVSRADID DVERGAVDLT WDPVDDAVAK LVDTEDTAIL HGWEEAGITG LSEASVHQPV QMPAELEQID DAVSGACNVL R LADVEGPY DLVLPQQLYT QVSETTDHGV PVVDHLTQLL SGGEVLWAPA ARCALVVSRR GGDSCLFLGR DVSIGYLSHD AQ TVTLYLE ESFTFRVHQP DAAVALV

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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 chloridesodium chloride
20.0 mMC4H11NO3Tris
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. / Pretreatment - Atmosphere: AIR
Details: The grid was glow discharged for 60 seconds at 5 mA.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
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

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 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: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 45000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Number grids imaged: 1 / Number real images: 975 / Average exposure time: 4.0 sec. / Average electron dose: 41.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
In silico model: An intial ab-initio model was created using cryoSPARC Ab-Initio Reconstruction.
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.3.1)
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 13581
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsA 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.
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 77.81 / Target criteria: Correlation coefficient
Output model

PDB-8dnl:
Acidipropionibacterium acidipropionici encapsulin in an open state at pH 7.5

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