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- PDB-9c67: cryoEM structure of CRISPR associated effector, CARF-Adenosine de... -

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Basic information

Entry
Database: PDB / ID: 9c67
TitlecryoEM structure of CRISPR associated effector, CARF-Adenosine deaminase 1, Cad1, in apo form
ComponentsAdenosine deaminase domain-containing protein
KeywordsANTIVIRAL PROTEIN / Antiphage defense / CRISPR / Deamination / CARF-Adenosine deaminase
Function / homology
Function and homology information


inosine biosynthetic process / adenosine catabolic process / adenosine deaminase activity
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 deaminase domain-containing protein
Similarity search - Component
Biological speciesBacteroidales bacterium (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsMajumder, P. / Patel, D.J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIHGM 129430, NIHGM 145888 United States
CitationJournal: Cell / Year: 2024
Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity.
Authors: Christian F Baca / Puja Majumder / James H Hickling / Linzhi Ye / Marianna Teplova / Sean F Brady / Dinshaw J Patel / Luciano A Marraffini /
Abstract: Type III CRISPR systems provide immunity against genetic invaders through the production of cyclic oligo-adenylate (cA) molecules that activate effector proteins that contain CRISPR-associated ...Type III CRISPR systems provide immunity against genetic invaders through the production of cyclic oligo-adenylate (cA) molecules that activate effector proteins that contain CRISPR-associated Rossman fold (CARF) domains. Here, we characterized the function and structure of an effector in which the CARF domain is fused to an adenosine deaminase domain, CRISPR-associated adenosine deaminase 1 (Cad1). We show that upon binding of cA or cA to its CARF domain, Cad1 converts ATP to ITP, both in vivo and in vitro. Cryoelectron microscopy (cryo-EM) structural studies on full-length Cad1 reveal an hexameric assembly composed of a trimer of dimers, with bound ATP at inter-domain sites required for activity and ATP/ITP within deaminase active sites. Upon synthesis of cA during phage infection, Cad1 activation leads to a growth arrest of the host that prevents viral propagation. Our findings reveal that CRISPR-Cas systems employ a wide range of molecular mechanisms beyond nucleic acid degradation to provide adaptive immunity in prokaryotes.
History
DepositionJun 7, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 30, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2024Group: 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_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Adenosine deaminase domain-containing protein
B: Adenosine deaminase domain-containing protein
C: Adenosine deaminase domain-containing protein
D: Adenosine deaminase domain-containing protein
E: Adenosine deaminase domain-containing protein
F: Adenosine deaminase domain-containing protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)403,73612
Polymers403,5906
Non-polymers1466
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
Adenosine deaminase domain-containing protein


Mass: 67264.945 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacteroidales bacterium (bacteria) / Gene: DCM62_02910 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A3C0QUR5
#2: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
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: apo Cad1 (CRISPR associated Adenosine deaminase 1) full length protein
Type: COMPLEX / Details: It forms a trimer of dimers / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.4 MDa / Experimental value: YES
Source (natural)Organism: Bacteriodale bacterium (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Details: 25 mM Hepes pH 8, 200 mM NaCl, 2 mM Beta-mercaptoethanol and 5 % glycerol
SpecimenConc.: 1.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Glow discharge 1 min, 15 mA 100 % humidity, wait time 12 s, 2.5 s blot time and 0 blot force at 4 degree C temperature
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN 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: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 53 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
12Cootmodel fitting
19PHENIX1.20.1_4487:model refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 110836
Details: Half maps were generated by Non-uniform refinement programme using cryoSparc
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00327362
ELECTRON MICROSCOPYf_angle_d0.74237058
ELECTRON MICROSCOPYf_dihedral_angle_d4.5683623
ELECTRON MICROSCOPYf_chiral_restr0.0444123
ELECTRON MICROSCOPYf_plane_restr0.0054799

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