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- PDB-9c6a: The CRISPR associated adenosine deaminase Cad1-CARF in the apo form -

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Basic information

Entry
Database: PDB / ID: 9c6a
TitleThe CRISPR associated adenosine deaminase Cad1-CARF in the apo form
ComponentsAdenosine deaminase domain-containing protein
KeywordsCYTOSOLIC PROTEIN / Type-III CRISPR defense system / CARF-effector protein / adaptive immunity / deamination defense strategy
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 speciesBacteroidales bacterium (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / 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)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

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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


Theoretical massNumber of molelcules
Total (without water)75,8634
Polymers75,8634
Non-polymers00
Water28816
1
A: Adenosine deaminase domain-containing protein
C: Adenosine deaminase domain-containing protein


Theoretical massNumber of molelcules
Total (without water)37,9312
Polymers37,9312
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2110 Å2
ΔGint-10 kcal/mol
Surface area16660 Å2
MethodPISA
2
B: Adenosine deaminase domain-containing protein
D: Adenosine deaminase domain-containing protein


Theoretical massNumber of molelcules
Total (without water)37,9312
Polymers37,9312
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2030 Å2
ΔGint-9 kcal/mol
Surface area16750 Å2
MethodPISA
Unit cell
Length a, b, c (Å)53.615, 96.801, 155.722
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein
Adenosine deaminase domain-containing protein


Mass: 18965.695 Da / Num. of mol.: 4
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: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 16 / 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: 2.66 Å3/Da / Density % sol: 53.82 % / Description: Needle
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8.5 / Details: 0.1 M Tris-HCl pH 8.5, 2 M ammonium sulfate

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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 16M / Detector: PIXEL / Date: Feb 4, 2024 / Details: Horizontal secondary source 2 stage focusing
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: 3.59→82.21 Å / Num. obs: 10011 / % possible obs: 100 % / Redundancy: 1.8 % / CC1/2: 0.923 / Rmerge(I) obs: 0.24 / Rpim(I) all: 0.24 / Rrim(I) all: 0.34 / Χ2: 0.77 / Net I/σ(I): 2 / Num. measured all: 18293
Reflection shellResolution: 3.59→3.93 Å / % possible obs: 100 % / Redundancy: 1.9 % / Rmerge(I) obs: 0.638 / Num. measured all: 4365 / Num. unique obs: 2326 / CC1/2: 0.503 / Rpim(I) all: 0.638 / Rrim(I) all: 0.902 / Χ2: 0.92 / Net I/σ(I) obs: 1.1

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Processing

Software
NameVersionClassification
PHENIX(1.20.1_4487: ???)refinement
Aimlessdata scaling
MOLREPCCP4i2phasing
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.6→82.21 Å / SU ML: 0.58 / Cross valid method: FREE R-VALUE / σ(F): 1.92 / Phase error: 31.15 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.317 522 5.28 %
Rwork0.2484 --
obs0.2521 9892 99.92 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 3.6→82.21 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5324 0 0 16 5340
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.003
X-RAY DIFFRACTIONf_angle_d0.617
X-RAY DIFFRACTIONf_dihedral_angle_d6.312700
X-RAY DIFFRACTIONf_chiral_restr0.045788
X-RAY DIFFRACTIONf_plane_restr0.005960
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.6-3.960.36111320.28092256X-RAY DIFFRACTION100
3.96-4.540.32351270.25242316X-RAY DIFFRACTION100
4.54-5.710.26911210.22132343X-RAY DIFFRACTION100
5.71-82.210.31941420.24852455X-RAY DIFFRACTION100

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