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- EMDB-63330: Cryo-electron tomogram of B. cereus CotE meshwork -

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Basic information

Entry
Database: EMDB / ID: EMD-63330
TitleCryo-electron tomogram of B. cereus CotE meshwork
Map dataCryo-electron tomography reconstruction of Bacillus cereus CotE protein meshwork
Sample
  • Complex: Bacillus cereus CotE protein meshwork assembled in vitro
KeywordsEndospore / Gram-positive bacteria / CotE protein / STRUCTURAL PROTEIN
Biological speciesBacillus cereus (bacteria)
Methodelectron tomography / cryo EM
AuthorsHa N-C / Lee D / Baek Y / Park M / Kim D / Byun K / Hyun J
Funding support Korea, Republic Of, 1 items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea)2022R1A2C109178313 Korea, Republic Of
CitationJournal: mBio / Year: 2025
Title: 3D meshwork architecture of the outer coat protein CotE: implications for bacterial endospore sporulation and germination.
Authors: Dukwon Lee / Yeongjin Baek / Migak Park / Doyeon Kim / Kyumi Byun / Jaekyung Hyun / Nam-Chul Ha /
Abstract: a Gram-positive aerobic bacterium commonly found in soil, food, and water, forms endospores that can withstand harsh environmental conditions. The endospores are encased in a protective spore coat ... a Gram-positive aerobic bacterium commonly found in soil, food, and water, forms endospores that can withstand harsh environmental conditions. The endospores are encased in a protective spore coat consisting of multiple layers of proteins, among which, CotE serves as a crucial morphogenetic protein within the outer coat. In this study, we observed that the homotrimeric CotE protein underwent further oligomerization induced by Ca and was subsequently dissociated by dipicolinic acid, a compound released from the spore core during germination. Through cryo-electron microscopy and tomography analyses of the Ca-induced CotE oligomer, combined with structural predictions and biochemical studies, we propose a three-dimensional meshwork organization facilitated by tryptophan-based interactions between CotE trimers. The resulting meshwork was organized in a defective diamond-like tetrahedral configuration. These insights enhance our understanding of how CotE contributes to endospore morphogenesis and germination through the rapid disassembly of these layers.
IMPORTANCE: Bacterial endospores are highly resilient structures that allow bacteria to survive extreme environmental conditions, making them a significant concern in food safety and healthcare. The ...IMPORTANCE: Bacterial endospores are highly resilient structures that allow bacteria to survive extreme environmental conditions, making them a significant concern in food safety and healthcare. The protein CotE plays a critical role in forming the protective outer coat of these endospores. Our research uncovers the three-dimensional meshwork architecture of CotE and reveals how it contributes to the structural integrity and rapid disassembly of endospores during germination. By understanding CotE's unique 3D structure and its interaction with other molecules, we gain valuable insights into how bacterial endospores are formed and how they can be effectively targeted for sterilization. This work not only advances our fundamental knowledge of bacterial endospore biology but also has potential applications in developing new strategies to combat bacterial contamination and improve sterilization techniques in the food and healthcare industries.
History
DepositionJan 31, 2025-
Header (metadata) releaseMar 12, 2025-
Map releaseMar 12, 2025-
UpdateMay 21, 2025-
Current statusMay 21, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_63330.png

Downloads & links

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Map

FileDownload / File: emd_63330.map.gz / Format: CCP4 / Size: 177.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-electron tomography reconstruction of Bacillus cereus CotE protein meshwork
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
10.98 Å/pix.
x 100 pix.
= 1098. Å		10.98 Å/pix.
x 682 pix.
= 7488.36 Å		10.98 Å/pix.
x 682 pix.
= 7488.36 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 10.98 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-7.6709185 - 16.912392000000001
Average (Standard dev.)0.43423375 (±0.3256292)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions682682100
Spacing682682100
CellA: 7488.36 Å / B: 7488.36 Å / C: 1098.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Bacillus cereus CotE protein meshwork assembled in vitro

EntireName: Bacillus cereus CotE protein meshwork assembled in vitro
Components
  • Complex: Bacillus cereus CotE protein meshwork assembled in vitro

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Supramolecule #1: Bacillus cereus CotE protein meshwork assembled in vitro

SupramoleculeName: Bacillus cereus CotE protein meshwork assembled in vitro
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Bacillus cereus (bacteria)
Molecular weightTheoretical: 200 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation state3D array

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Sample preparation

Concentration2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTris-HCltris(hydroxymethyl)aminomethane
0.005 mMCaCl2calcium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was semi-regular 3D meshwork composed of CotE protein
Cryo protectantN
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: AURION / Diameter: 10 nm

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Electron microscopy

MicroscopeTFS KRIOS
TemperatureMin: 77.0 K / Max: 98.0 K
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 41 / Average exposure time: 0.92 sec. / Average electron dose: 2.9 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 57000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: SIMULTANEOUS ITERATIVE (SIRT)
Software - details: Tomograms were post-processed using IsoNet (v0.2)
Number images used: 41
CTF correctionType: NONE

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