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- PDB-9l9d: Bacillus subtilis endospore crust protein CgeA -

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Basic information

Entry
Database: PDB / ID: 9l9d
TitleBacillus subtilis endospore crust protein CgeA
ComponentsSpore crust protein CgeA
KeywordsSTRUCTURAL PROTEIN / Glycoprotein / Sporulation / Bacillus subtilis
Function / homologySpore crust protein CgeA
Function and homology information
Biological speciesBacillus subtilis subsp. subtilis str. 168 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.05 Å
AuthorsPark, M. / Kim, D. / Baek, Y. / Hyun, J. / Ha, N.C.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: J Microbiol / Year: 2025
Title: Cryo-EM structure of the glycosylated protein CgeA in the crust of Bacillus subtilis endospores.
Authors: Migak Park / Doyeon Kim / Yeongjin Baek / Eunbyul Jo / Jaekyung Hyun / Nam-Chul Ha /
Abstract: The Bacillus subtilis spore crust is an exceptionally robust proteinaceous layer that protects spores under extreme environmental conditions. Among its key components, CgeA, a glycosylation- ...The Bacillus subtilis spore crust is an exceptionally robust proteinaceous layer that protects spores under extreme environmental conditions. Among its key components, CgeA, a glycosylation-associated protein, plays a critical role in modifying crust properties through its glycosylated moiety, enhancing spore dispersal in aqueous environments. In this study, we present the high-resolution cryo-electron microscopy structure of the core region of CgeA at 3.05 Å resolution, revealing a doughnut-like hexameric assembly. The N-terminal regions are disordered, whereas the C-terminal region forms the core of the hexamer. Although the loop containing Thr112 was not resolved in the density map, its location can be inferred from surrounding residues, suggesting that Thr112 is situated on the exposed surface of the hexamer. On the opposite face, a distinct electrostatic pattern is observed, featuring a negatively charged central pore and a positively charged outer surface. Modeling and biochemical studies with the putative glycosyltransferase CgeB provide insights into how the glycosyl group is transferred to Thr112. This study offers a molecular-level understanding of the assembly, glycosylation, and environmental adaptability of the B. subtilis spore crust, with valuable implications for controlling spore formation in industrial applications.
History
DepositionDec 30, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 12, 2025Provider: repository / Type: Initial release
Revision 1.0Nov 12, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Nov 12, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Nov 12, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Nov 12, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Nov 12, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Spore crust protein CgeA
B: Spore crust protein CgeA
C: Spore crust protein CgeA
D: Spore crust protein CgeA
E: Spore crust protein CgeA
F: Spore crust protein CgeA


Theoretical massNumber of molelcules
Total (without water)87,0216
Polymers87,0216
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, Cryo-EM analysis revealed that the particles form a hexamer., light scattering, MALS analysis revealed that the protein forms a hexamer.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Spore crust protein CgeA


Mass: 14503.417 Da / Num. of mol.: 6 / Mutation: A42C,D97C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Strain: 168 / Gene: cgeA, cgeAA, BSU19780 / Plasmid: pProEXHTA / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P42089
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

ComponentName: Hexameric complex of Bacillus subtilis endospore crust protein CgeA
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.084 MDa / Experimental value: YES
Source (natural)Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Strain: 168
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pProEXHTA
Buffer solutionpH: 8 / Details: 20 mM Tris-HCl, 150 mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris bufferTris-HCl1
2150 mMSodium chlorideNaCl1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 1700 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 63.6 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 10593

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.5.1particle selection
7UCSF ChimeraXmodel fitting
8Cootmodel fitting
10PHENIXmodel refinement
14cryoSPARC4.5.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 88923 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: Initial local fitting was done using Coot and Chimera and then PHENIX was used for flexible fitting
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementHighest resolution: 3.05 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0043108
ELECTRON MICROSCOPYf_angle_d0.664242
ELECTRON MICROSCOPYf_dihedral_angle_d4.757412
ELECTRON MICROSCOPYf_chiral_restr0.051534
ELECTRON MICROSCOPYf_plane_restr0.004528

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