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- PDB-9c68: The CRISPR associated CARF-adenosine deaminase Cad1-CARF in the c... -

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Basic information

Entry
Database: PDB / ID: 9c68
TitleThe CRISPR associated CARF-adenosine deaminase Cad1-CARF in the cA6 bound form
Components
  • Adenosine deaminase domain-containing protein
  • RNA (5'-R(P*AP*AP*AP*AP*AP*A)-3')
KeywordsAntiviral Protein/RNA / Type-III CRISPR defense system / CARF-effector protein / adaptive immunity / deamination defense strategy / cyclic Hexa-adenylate / Antiviral Protein-RNA complex
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
RNA / Adenosine deaminase domain-containing protein
Similarity search - Component
Biological speciesBacteroidales bacterium (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.82 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Adenosine deaminase domain-containing protein
B: Adenosine deaminase domain-containing protein
C: RNA (5'-R(P*AP*AP*AP*AP*AP*A)-3')


Theoretical massNumber of molelcules
Total (without water)44,0693
Polymers44,0693
Non-polymers00
Water88349
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, SECMALS
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4960 Å2
ΔGint-6 kcal/mol
Surface area14560 Å2
MethodPISA
Unit cell
Length a, b, c (Å)85.925, 85.925, 188.721
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number180
Space group name H-MP6222
Components on special symmetry positions
IDModelComponents
11A-216-

HOH

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Components

#1: Protein Adenosine deaminase domain-containing protein


Mass: 21069.125 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 RNA (5'-R(P*AP*AP*AP*AP*AP*A)-3')


Mass: 1930.277 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.: 49 / 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: 2.28 Å3/Da / Density % sol: 46.1 % / Description: small diamond
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 4.5 / Details: 0.1 M sodium acetate pH 4.5, 35 % (v/v) MPD

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: NSLS-II / Beamline: 17-ID-2 / Wavelength: 0.9793 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Feb 4, 2024 / Details: KB bimorph mirrors
RadiationMonochromator: Si(111) DCM / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9793 Å / Relative weight: 1
ReflectionResolution: 1.82→74.41 Å / Num. obs: 37752 / % possible obs: 100 % / Redundancy: 1.8 % / CC1/2: 0.999 / Rmerge(I) obs: 0.038 / Rpim(I) all: 0.038 / Rrim(I) all: 0.053 / Χ2: 0.92 / Net I/σ(I): 9.5 / Num. measured all: 69781
Reflection shellResolution: 1.82→1.86 Å / % possible obs: 99.9 % / Redundancy: 1.9 % / Rmerge(I) obs: 2.017 / Num. measured all: 4137 / Num. unique obs: 2181 / CC1/2: 0.421 / Rpim(I) all: 2.017 / Rrim(I) all: 2.853 / Χ2: 0.79 / Net I/σ(I) obs: 0.4

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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: 1.82→42.96 Å / SU ML: 0.32 / Cross valid method: FREE R-VALUE / σ(F): 1.89 / Phase error: 34.89 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.3123 1902 5.05 %
Rwork0.2681 --
obs0.2703 37688 99.97 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.82→42.96 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2628 132 0 49 2809
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01
X-RAY DIFFRACTIONf_angle_d1.426
X-RAY DIFFRACTIONf_dihedral_angle_d10.06414
X-RAY DIFFRACTIONf_chiral_restr0.078421
X-RAY DIFFRACTIONf_plane_restr0.009482
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.82-1.870.38791250.38712510X-RAY DIFFRACTION100
1.87-1.920.36471220.33872484X-RAY DIFFRACTION100
1.92-1.970.32361350.33192494X-RAY DIFFRACTION100
1.97-2.040.34991200.3372536X-RAY DIFFRACTION100
2.04-2.110.37131550.31462479X-RAY DIFFRACTION100
2.11-2.190.37721300.33012518X-RAY DIFFRACTION100
2.2-2.290.35851220.31042534X-RAY DIFFRACTION100
2.29-2.420.35691380.31692532X-RAY DIFFRACTION100
2.42-2.570.38031600.31552513X-RAY DIFFRACTION100
2.57-2.770.32281420.31012558X-RAY DIFFRACTION100
2.77-3.040.34941290.29622571X-RAY DIFFRACTION100
3.04-3.480.30181340.27212593X-RAY DIFFRACTION100
3.48-4.390.29721570.22492622X-RAY DIFFRACTION100
4.39-42.960.26331330.23142842X-RAY DIFFRACTION100

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