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- PDB-9ofb: CryoEM structure of Cad1 bound with cA4 and ATP, symmetry expande... -

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Basic information

Entry
Database: PDB / ID: 9ofb
TitleCryoEM structure of Cad1 bound with cA4 and ATP, symmetry expanded dimer refined against a composite map
Components
  • CRISPR-associated ring nuclease and adenosine deaminase, subunit A
  • cA4 cleavage product, A2>P
KeywordsIMMUNE SYSTEM / HYDROLASE / CRISPR-associated Rossman-fold (CARF) / ATP deaminase / CRISPR immunity / cooperative activation
Function / homology
Function and homology information


inosine biosynthetic process / adenosine deaminase / hypoxanthine salvage / adenosine deaminase activity / adenosine catabolic process / cytosol
Similarity search - Function
CRISPR-assoc protein, NE0113/Csx13 / CRISPR-associated protein NE0113 (Cas_NE0113) / Adenosine deaminase domain / Adenosine deaminase / Adenosine/adenine deaminase / Metal-dependent hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / RNA / adenosine deaminase
Similarity search - Component
Biological speciesThermoanaerobaculum aquaticum (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.68 Å
AuthorsZhao, Y. / Li, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM152081 United States
CitationJournal: EMBO J / Year: 2025
Title: The twist-and-squeeze activation of CARF-fused adenosine deaminase by cyclic oligoadenylates.
Authors: Charlisa Whyms / Yu Zhao / Doreen Addo-Yobo / Huan He / Arthur Carl Whittington / Despoina Trasanidou / Carl Raymund P Salazar / Raymond H J Staals / Hong Li /
Abstract: The recently identified CARF (CRISPR-associated Rossman-fold) family of proteins play a critical role in prokaryotic defense, mediating cOA (cyclic oligoadenylate)-stimulated ancillary immune ...The recently identified CARF (CRISPR-associated Rossman-fold) family of proteins play a critical role in prokaryotic defense, mediating cOA (cyclic oligoadenylate)-stimulated ancillary immune responses in the type III CRISPR-Cas systems. Whereas most previously characterized CARF proteins contain nucleic acids or protein degradation effectors, a subset of the family, including the CARF-fused adenosine deaminase (ADA) (Cad1), has recently been shown to convert ATP to ITP. The enzymatic mechanism and the activation process of Cad1, however, remain incompletely understood. Here we present biochemical and structural analyses of a ring nuclease Cad1, revealing its substrate binding specificity and a sequential activation process by cOAs. Despite an overall structural similarity to canonical ADA enzymes, the ADA domain of Cad1 possesses unique structural features that confer a specificity for ATP. Supported by mutational analysis, our structural work demonstrates an allosteric link between the cOA-binding CARF and the ADA domain through a protein network within the hexameric enzyme assembly. Binding of a cA4 molecule to paired CARF domains induces a twisting of the linked ADA domains around one another, which remodels their active sites and alters interactions with neighboring ADA domains, thereby driving a sequential conformational activation mechanism.
History
DepositionApr 29, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 20, 2025Provider: repository / Type: Initial release
Revision 1.0Aug 20, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Aug 20, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Aug 20, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Oct 29, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated ring nuclease and adenosine deaminase, subunit A
B: CRISPR-associated ring nuclease and adenosine deaminase, subunit A
C: cA4 cleavage product, A2>P
D: cA4 cleavage product, A2>P
hetero molecules


Theoretical massNumber of molelcules
Total (without water)141,59212
Polymers140,3494
Non-polymers1,2428
Water1086
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein / RNA chain , 2 types, 4 molecules ABCD

#1: Protein CRISPR-associated ring nuclease and adenosine deaminase, subunit A


Mass: 69499.133 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermoanaerobaculum aquaticum (bacteria)
Gene: EG19_07865 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A062XY19, adenosine deaminase
#2: RNA chain cA4 cleavage product, A2>P


Mass: 675.419 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Thermoanaerobaculum aquaticum (bacteria)

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Non-polymers , 4 types, 14 molecules

#3: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
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: Cad1 / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Thermoanaerobaculum aquaticum (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 defocus max: 2500 nm / Nominal defocus min: 820 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 257900 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 101.69 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00379910
ELECTRON MICROSCOPYf_angle_d0.553113540
ELECTRON MICROSCOPYf_chiral_restr0.04081489
ELECTRON MICROSCOPYf_plane_restr0.00461755
ELECTRON MICROSCOPYf_dihedral_angle_d5.25311357

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