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- PDB-9eka: CRISPR-associated deaminase Cad1 in Apo form -

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Basic information

Entry
Database: PDB / ID: 9eka
TitleCRISPR-associated deaminase Cad1 in Apo form
ComponentsCRISPR-associated ring nuclease and adenosine deaminase, subunit A
KeywordsHYDROLASE / CRISPR-associated CARF ring nuclease / Cad1 / CRISPR-associated adenosine deaminase / S-adenosylmethionine / SIGNALING PROTEIN
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
Biological speciesThermoanaerobaculum aquaticum (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsZhao, Y. / Whyms, C.T. / Li, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM152081 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
DepositionDec 2, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 7, 2026Provider: repository / Type: Initial release
Revision 1.0Jan 7, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jan 7, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jan 7, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jan 7, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jan 7, 2026Data 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
B: CRISPR-associated ring nuclease and adenosine deaminase, subunit A
A: CRISPR-associated ring nuclease and adenosine deaminase, subunit A
F: CRISPR-associated ring nuclease and adenosine deaminase, subunit A
E: CRISPR-associated ring nuclease and adenosine deaminase, subunit A
H: CRISPR-associated ring nuclease and adenosine deaminase, subunit A
G: CRISPR-associated ring nuclease and adenosine deaminase, subunit A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)417,38712
Polymers416,9956
Non-polymers3926
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
CRISPR-associated ring nuclease and adenosine deaminase, subunit A


Mass: 69499.133 Da / Num. of mol.: 6
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: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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: protein-cA4 complex / Type: COMPLEX / Entity ID: #1 / 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 magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21.2_5419 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 15816 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 201.01 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.027429304
ELECTRON MICROSCOPYf_angle_d1.627239921
ELECTRON MICROSCOPYf_chiral_restr0.08854407
ELECTRON MICROSCOPYf_plane_restr0.01395262
ELECTRON MICROSCOPYf_dihedral_angle_d5.10983999

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