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- PDB-9c69: The CRISPR associated CARF-adenosine deaminase, Cad1-CARF in the ... -

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Basic information

Entry
Database: PDB / ID: 9c69
TitleThe CRISPR associated CARF-adenosine deaminase, Cad1-CARF in the cA4 bound form
Components
  • AAAA
  • Adenosine deaminase domain-containing protein
KeywordsAntiviral Protein/RNA / Type-III CRISPR defense system / CARF-effector protein / adaptive immunity / deamination defense strategy / CYTOSOLIC PROTEIN / cyclic Tetra-adenylate / antiphage defense / Antiviral Protein-RNA complex
Function / homology
Function and homology information


inosine biosynthetic process / adenosine deaminase / hypoxanthine salvage / adenosine catabolic process / adenosine deaminase activity / 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
RNA / adenosine deaminase
Similarity search - Component
Biological speciesBacteroidales bacterium (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.4 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)NIHGM129430, NIHGM145888 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: Database references / Category: citation / citation_author
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
Revision 1.2Dec 25, 2024Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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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: AAAA


Theoretical massNumber of molelcules
Total (without water)39,2033
Polymers39,2033
Non-polymers00
Water50428
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, SECMALS
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3660 Å2
ΔGint-15 kcal/mol
Surface area15590 Å2
MethodPISA
Unit cell
Length a, b, c (Å)189.308, 189.308, 189.308
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number211
Space group name H-MI432

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Components

#1: Protein Adenosine deaminase domain-containing protein


Mass: 18965.695 Da / Num. of mol.: 2
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: RNA chain AAAA


Mass: 1271.866 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Bacteroidales bacterium (bacteria)
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 28 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.3 Å3/Da / Density % sol: 63.5 % / Description: diamond
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 5
Details: 1 M lithium chloride, 0.1 M citric acid pH 5, 10 % (w/v) PEG 6000

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Data collection

DiffractionMean temperature: 293 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: NSLS-II / Beamline: 19-ID / Wavelength: 0.9793 Å
DetectorType: DECTRIS EIGER2 XE 9M / Detector: PIXEL / Date: Feb 4, 2024
RadiationMonochromator: DCM with sagittal focusing / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9793 Å / Relative weight: 1
ReflectionResolution: 2.4→47.33 Å / Num. obs: 22654 / % possible obs: 99 % / Redundancy: 1 % / CC1/2: 1 / Rmerge(I) obs: 0.099 / Χ2: 0 / Net I/σ(I): 40.5
Reflection shellResolution: 2.4→2.49 Å / Redundancy: 1 % / Rmerge(I) obs: 0.537 / Num. unique obs: 2367 / CC1/2: 0.98 / Χ2: 0

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Processing

Software
NameVersionClassification
PHENIX(1.20.1_4487: ???)refinement
Aimlessdata scaling
MOLREPphasing
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.4→47.33 Å / SU ML: 0.31 / Cross valid method: FREE R-VALUE / σ(F): 1.95 / Phase error: 24.05 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2519 1176 5.19 %
Rwork0.2015 --
obs0.2041 22654 98.89 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.4→47.33 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2634 88 0 28 2750
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.009
X-RAY DIFFRACTIONf_angle_d1.065
X-RAY DIFFRACTIONf_dihedral_angle_d10.025395
X-RAY DIFFRACTIONf_chiral_restr0.059414
X-RAY DIFFRACTIONf_plane_restr0.008484
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.4-2.510.33341380.2452651X-RAY DIFFRACTION100
2.51-2.640.30991540.22472667X-RAY DIFFRACTION100
2.64-2.810.26871540.22962670X-RAY DIFFRACTION100
2.81-3.020.2931320.25492682X-RAY DIFFRACTION100
3.02-3.330.2811660.23862675X-RAY DIFFRACTION100
3.33-3.810.27441500.19722716X-RAY DIFFRACTION100
3.81-4.80.19821340.16842752X-RAY DIFFRACTION100
4.8-47.330.21061480.17112665X-RAY DIFFRACTION92

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