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- PDB-9mue: Cryo-EM structure of CRISPR-associated cA4 bound Cat1 Pentagonal ... -

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Basic information

Entry
Database: PDB / ID: 9mue
TitleCryo-EM structure of CRISPR-associated cA4 bound Cat1 Pentagonal filament assembly in the presence of NAD (ADPR modelled)
Components
  • Cat1 (CRISPR associated TIR 1) pentagonal filament assembly
  • RNA (5'-R(P*AP*AP*AP*A)-3')
KeywordsANTIVIRAL PROTEIN / CRISPR / antiphage defense / adaptive immunity / Filament / CARF / TIR / NAD / ADPR / NAM
Function / homologyChem-AR6 / RNA
Function and homology information
Biological speciesbacterium (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsMajumder, P. / Patel, D.J.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)GM129430 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)GM145888 United States
CitationJournal: Science / Year: 2025
Title: Cat1 forms filament networks to degrade NAD during the type III CRISPR-Cas antiviral response.
Authors: Christian F Baca / Puja Majumder / James H Hickling / Dinshaw J Patel / Luciano A Marraffini /
Abstract: Type III CRISPR-Cas systems defend against viral infection in prokaryotes by using an RNA-guided complex that recognizes foreign transcripts and synthesizes cyclic oligoadenylate (cOA) messengers to ...Type III CRISPR-Cas systems defend against viral infection in prokaryotes by using an RNA-guided complex that recognizes foreign transcripts and synthesizes cyclic oligoadenylate (cOA) messengers to activate CRISPR-associated Rossmann-fold (CARF) immune effectors. In this study, we investigated a protein containing a CARF domain-fused Toll/interleukin-1 receptor (TIR) domain, Cat1. We found that Cat1 provides immunity by cleaving and depleting oxidized nicotinamide adenine dinucleotide (NAD) molecules from the infected host, inducing a growth arrest that prevents viral propagation. Cat1 forms dimers that stack upon each other to generate long filaments that are maintained by bound cOA ligands, with stacked TIR domains forming the NAD cleavage catalytic sites. Furthermore, Cat1 filaments assemble into distinct trigonal and pentagonal networks that enhance NAD degradation. Cat1 presents an unprecedented chemistry and higher-order protein assembly for the CRISPR-Cas response.
History
DepositionJan 13, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 16, 2025Provider: repository / Type: Initial release
Revision 1.1Apr 23, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.2Jun 25, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Cat1 (CRISPR associated TIR 1) pentagonal filament assembly
a: RNA (5'-R(P*AP*AP*AP*A)-3')
b: RNA (5'-R(P*AP*AP*AP*A)-3')
B: Cat1 (CRISPR associated TIR 1) pentagonal filament assembly
C: Cat1 (CRISPR associated TIR 1) pentagonal filament assembly
D: Cat1 (CRISPR associated TIR 1) pentagonal filament assembly
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,2658
Polymers123,1466
Non-polymers1,1192
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Cat1 (CRISPR associated TIR 1) pentagonal filament assembly


Mass: 30150.592 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) bacterium (bacteria) / Production host: Escherichia coli (E. coli)
#2: RNA chain RNA (5'-R(P*AP*AP*AP*A)-3')


Mass: 1271.866 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) bacterium (bacteria)
#3: Chemical ChemComp-AR6 / [(2R,3S,4R,5R)-5-(6-AMINOPURIN-9-YL)-3,4-DIHYDROXY-OXOLAN-2-YL]METHYL[HYDROXY-[[(2R,3S,4R,5S)-3,4,5-TRIHYDROXYOXOLAN-2-YL]METHOXY]PHOSPHORYL] HYDROGEN PHOSPHATE / Adenosine-5-Diphosphoribose


Mass: 559.316 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C15H23N5O14P2 / 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: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Antiviral protein Cat1 - Cyclic Tetra-Adenylate Complex NAD substrate was mixed with the sample.
Type: COMPLEX
Details: Cat1 protein forms filament structure upon recognition of cA4, Cyclic Tetra-Adenylate ligand. NAD substrate was mixed with the sample.
Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: bacterium (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6
SpecimenConc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: liquid-nitrogen-cooled liquid ethane was used as the cryogen.

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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 defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 57.32 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
7Cootmodel fitting
9PHENIX1.20.1_4487:model refinement
CTF correctionType: NONE
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 152750 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0048678
ELECTRON MICROSCOPYf_angle_d0.72611830
ELECTRON MICROSCOPYf_dihedral_angle_d8.9711300
ELECTRON MICROSCOPYf_chiral_restr0.0461324
ELECTRON MICROSCOPYf_plane_restr0.0071458

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