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- PDB-9lgh: Cryo-EM structure of CotVW filament, bacillus subtilis endospore ... -

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Basic information

Entry
Database: PDB / ID: 9lgh
TitleCryo-EM structure of CotVW filament, bacillus subtilis endospore protein
Components
  • Spore coat protein V
  • Spore coat protein W
KeywordsSTRUCTURAL PROTEIN / bacillus / spore protein / complex / filament / helical / PROTEIN FIBRIL
Function / homologySpore coat protein X/V / Spore Coat Protein X and V domain / spore wall / sporulation resulting in formation of a cellular spore / Spore coat protein V / Spore coat protein W
Function and homology information
Biological speciesBacillus subtilis subsp. subtilis str. 168 (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.32 Å
AuthorsJo, E. / Kim, D. / Baek, Y. / Ha, N.-C.
Funding support Korea, Republic Of, 1items
OrganizationGrant numberCountry
Not funded Korea, Republic Of
CitationJournal: J Biol Chem / Year: 2025
Title: Filamentous structure of the CotVW complex, the crust proteins of the Bacillus subtilis endospore.
Authors: Eunbyul Jo / Doyeon Kim / Yeongjin Baek / Migak Park / Hyojeong Lee / Nam-Chul Ha /
Abstract: The endospores of Bacillus subtilis are encased in a multilayered protective structure comprising core, cortex, inner and outer coats, and an outermost crust. Among the proteins required for crust ...The endospores of Bacillus subtilis are encased in a multilayered protective structure comprising core, cortex, inner and outer coats, and an outermost crust. Among the proteins required for crust formation, CotV and CotW are unique to B. subtilis and are hypothesized to be instrumental in maintaining spore surface integrity. However, their structural organization and functional mechanisms remain unclear. This study determined the cryo-EM structure of the CotVW complex and revealed its filamentous helical architecture. Structural analysis showed that CotVW possesses a negatively charged surface that enables pH-dependent binding interactions. Specifically, at pH 6.0, CotVW engages in electrostatic interactions with histidine and positively charged residues, suggesting a potential regulatory mechanism influenced by the environmental pH. Our results elucidate the molecular basis of CotVW function in B. subtilis spore crust formation, highlighting its role in spore surface organization. This study advances our understanding of the spore coat architecture and may inform future research on bacterial spore resilience and structural adaptation.
History
DepositionJan 10, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 24, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
V: Spore coat protein V
W: Spore coat protein W


Theoretical massNumber of molelcules
Total (without water)28,5432
Polymers28,5432
Non-polymers00
Water00
1
V: Spore coat protein V
W: Spore coat protein W

V: Spore coat protein V
W: Spore coat protein W


Theoretical massNumber of molelcules
Total (without water)57,0854
Polymers57,0854
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
point symmetry operation1

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Components

#1: Protein Spore coat protein V


Mass: 16018.232 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: This sequence includes an expression tag (MGSSHHHHHHSQDP) and an additional tyrosine residue was arbitrarily added at the C-terminus for purification convenience.
Source: (gene. exp.) Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Gene: cotV, BSU11780 / Production host: Escherichia coli (E. coli) / References: UniProt: Q08309
#2: Protein Spore coat protein W


Mass: 12524.431 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: An additional tyrosine residue was arbitrarily added at the C-terminus for purification convenience.
Source: (gene. exp.) Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Gene: cotW, BSU11770 / Production host: Escherichia coli (E. coli) / References: UniProt: Q08310
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Helical filament of Bacillus subtilis endospore protein CotVW
Type: COMPLEX
Details: The CotVW filament is composed of repeating heterodimeric units of CotV and CotW.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 26.5 kDa/nm / Experimental value: NO
Source (natural)Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMTris(hydroxymethyl)aminomethane hydrochlorideTris-HCl1
SpecimenEmbedding 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
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen 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: 96000 X / Nominal defocus max: 1700 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4742

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.6.0particle selectionauto filament picking
4cryoSPARC4.6.0CTF correctionpatch ctf
7Coot0.9.8model fitting
9PHENIX1.21model refinement
10cryoSPARC4.6.0initial Euler assignment
11cryoSPARC4.6.0final Euler assignment
12cryoSPARC4.6.0classification
13cryoSPARC4.6.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 153.09 ° / Axial rise/subunit: 9.52 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 150717
3D reconstructionResolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 137931 / Symmetry type: HELICAL
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingDetails: An initial model was generated using ModelAngelo. / Source name: Other / Type: in silico model
RefinementHighest resolution: 3.32 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0021643
ELECTRON MICROSCOPYf_angle_d0.572208
ELECTRON MICROSCOPYf_dihedral_angle_d3.608209
ELECTRON MICROSCOPYf_chiral_restr0.041277
ELECTRON MICROSCOPYf_plane_restr0.002278

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