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- PDB-9eyi: Cryo-EM structure of SAVED-Lon protease CCaCalpL filament bound to A4p -

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Basic information

Entry
Database: PDB / ID: 9eyi
TitleCryo-EM structure of SAVED-Lon protease CCaCalpL filament bound to A4p
Components
  • A4p (5'-R(*AP*AP*AP*AP)-3')
  • SMODS-associated and fused to various effectors domain-containing protein
KeywordsHYDROLASE / LON PROTEASE / SAVED DOMAIN / CRISPR
Function / homologySMODS-associated and fused to various effectors / SMODS-associated and fused to various effectors sensor domain / RNA / SMODS-associated and fused to various effectors domain-containing protein
Function and homology information
Biological speciesCandidatus Cloacimonas acidaminovorans (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.75 Å
AuthorsTamulaitiene, G. / Sasnauskas, G. / Smalakyte, D. / Tamulaitis, G.
Funding supportLithuania, 1items
OrganizationGrant numberCountry
Research Council of LithuaniaS-MIP-22-9Lithuania
CitationJournal: Mol Cell / Year: 2024
Title: Filament formation activates protease and ring nuclease activities of CRISPR Lon-SAVED.
Authors: Dalia Smalakyte / Audrone Ruksenaite / Giedrius Sasnauskas / Giedre Tamulaitiene / Gintautas Tamulaitis
Abstract: To combat phage infection, type III CRISPR-Cas systems utilize cyclic oligoadenylates (cA) signaling to activate various auxiliary effectors, including the CRISPR-associated Lon-SAVED protease CalpL, ...To combat phage infection, type III CRISPR-Cas systems utilize cyclic oligoadenylates (cA) signaling to activate various auxiliary effectors, including the CRISPR-associated Lon-SAVED protease CalpL, which forms a tripartite effector system together with an anti-σ factor, CalpT, and an ECF-like σ factor, CalpS. Here, we report the characterization of the Candidatus Cloacimonas acidaminovorans CalpL-CalpT-CalpS. We demonstrate that cA binding triggers CalpL filament formation and activates it to cleave CalpT within the CalpT-CalpS dimer. This cleavage exposes the CalpT C-degron, which targets it for further degradation by cellular proteases. Consequently, CalpS is released to bind to RNA polymerase, causing growth arrest in E. coli. Furthermore, the CalpL-CalpT-CalpS system is regulated by the SAVED domain of CalpL, which is a ring nuclease that cleaves cA in a sequential three-step mechanism. These findings provide key mechanistic details for the activation, proteolytic events, and regulation of the signaling cascade in the type III CRISPR-Cas immunity.
History
DepositionApr 9, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 2, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2024Group: Data collection / Database references / Structure summary
Category: citation / em_admin / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: SMODS-associated and fused to various effectors domain-containing protein
B: SMODS-associated and fused to various effectors domain-containing protein
C: SMODS-associated and fused to various effectors domain-containing protein
D: A4p (5'-R(*AP*AP*AP*AP)-3')
E: A4p (5'-R(*AP*AP*AP*AP)-3')


Theoretical massNumber of molelcules
Total (without water)192,5865
Polymers192,5865
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein SMODS-associated and fused to various effectors domain-containing protein / SAVED-Lon protease CcaCalpL


Mass: 63294.172 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Candidatus Cloacimonas acidaminovorans (bacteria)
Strain: Evry / Gene: CLOAM0848 / Plasmid: pBAD/HisA / Production host: Escherichia coli (E. coli) / Strain (production host): DH10B / References: UniProt: B0VHB4, endopeptidase La
#2: RNA chain A4p (5'-R(*AP*AP*AP*AP)-3')


Mass: 1351.846 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Filament fragment of A4p bound CCaCalpLCOMPLEXall0MULTIPLE SOURCES
2CCaCalpLCOMPLEX#11RECOMBINANT
3A4pCOMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Candidatus Cloacimonas acidaminovorans (bacteria)456827
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33synthetic construct (others)32630
Buffer solutionpH: 7.5
SpecimenConc.: 1 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

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 92000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 30 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
13cryoSPARCv4.4.13D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 2.75 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 175826 / Symmetry type: POINT

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