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- EMDB-48630: Cryo-EM structure of CRISPR-associated cA4 bound Cat1 Pentagonal ... -

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Basic information

Entry
Database: EMDB / ID: EMD-48630
TitleCryo-EM structure of CRISPR-associated cA4 bound Cat1 Pentagonal filament assembly in the presence of NAD (ADPR modelled)
Map dataMain cryo-EM map Z-flipped
Sample
  • Complex: Antiviral protein Cat1 - Cyclic Tetra-Adenylate Complex NAD substrate was mixed with the sample.
    • Protein or peptide: Cat1 (CRISPR associated TIR 1) pentagonal filament assembly
    • RNA: RNA (5'-R(P*AP*AP*AP*A)-3')
  • Ligand: [(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
KeywordsCRISPR / antiphage defense / adaptive immunity / Filament / CARF / TIR / NAD / ADPR / NAM / ANTIVIRAL PROTEIN
Biological speciesbacterium (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsMajumder P / Patel DJ
Funding support United States, 2 items
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, 2025-
Header (metadata) releaseApr 16, 2025-
Map releaseApr 16, 2025-
UpdateJun 25, 2025-
Current statusJun 25, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48630.png

Downloads & links

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Map

FileDownload / File: emd_48630.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain cryo-EM map Z-flipped
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 512 pix.
= 438.272 Å		0.86 Å/pix.
x 512 pix.
= 438.272 Å		0.86 Å/pix.
x 512 pix.
= 438.272 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.856 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-0.7297133 - 1.1902366
Average (Standard dev.)0.005066168 (±0.050016254)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 438.272 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: cryo-EM map half map A Z-flipped

Fileemd_48630_half_map_1.map
Annotationcryo-EM map half map A Z-flipped
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: cryo-EM map half map B Z-flipped

Fileemd_48630_half_map_2.map
Annotationcryo-EM map half map B Z-flipped
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Antiviral protein Cat1 - Cyclic Tetra-Adenylate Complex NAD subst...

EntireName: Antiviral protein Cat1 - Cyclic Tetra-Adenylate Complex NAD substrate was mixed with the sample.
Components
  • Complex: Antiviral protein Cat1 - Cyclic Tetra-Adenylate Complex NAD substrate was mixed with the sample.
    • Protein or peptide: Cat1 (CRISPR associated TIR 1) pentagonal filament assembly
    • RNA: RNA (5'-R(P*AP*AP*AP*A)-3')
  • Ligand: [(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

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Supramolecule #1: Antiviral protein Cat1 - Cyclic Tetra-Adenylate Complex NAD subst...

SupramoleculeName: Antiviral protein Cat1 - Cyclic Tetra-Adenylate Complex NAD substrate was mixed with the sample.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Cat1 protein forms filament structure upon recognition of cA4, Cyclic Tetra-Adenylate ligand. NAD substrate was mixed with the sample.
Source (natural)Organism: bacterium (bacteria)

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

MacromoleculeName: Cat1 (CRISPR associated TIR 1) pentagonal filament assembly
type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: bacterium (bacteria)
Molecular weightTheoretical: 30.150592 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPQAFFSHNN KDKKIVLEVL EHLRQSLVAT WIDQQSIPGG GSLIQQIIAG ISKSQYFLAF LSNEYLKSDW CWDELEQAYA LHQKGKVKI IPILLTNRAQ LDLNALTDAR RNFLESILTR LKYVEFDPHN MTRSLGSVAE ALWQNEAVRF EPIRMIKVNG T ELQVVEFK ...String:
MPQAFFSHNN KDKKIVLEVL EHLRQSLVAT WIDQQSIPGG GSLIQQIIAG ISKSQYFLAF LSNEYLKSDW CWDELEQAYA LHQKGKVKI IPILLTNRAQ LDLNALTDAR RNFLESILTR LKYVEFDPHN MTRSLGSVAE ALWQNEAVRF EPIRMIKVNG T ELQVVEFK IPGSNLPVDF LHHWDLKIED FIATSPNEQK PVKFDVPVAL YGPGPNWLYA FLTLPFKNRN TVFVFNSRTS EY ICVYSKS AGLAPGMVLK GHHHHH

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Macromolecule #2: RNA (5'-R(P*AP*AP*AP*A)-3')

MacromoleculeName: RNA (5'-R(P*AP*AP*AP*A)-3') / type: rna / ID: 2 / Number of copies: 2
Source (natural)Organism: bacterium (bacteria)
Molecular weightTheoretical: 1.271866 KDa
SequenceString:
AAAA

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Macromolecule #3: [(2R,3S,4R,5R)-5-(6-AMINOPURIN-9-YL)-3,4-DIHYDROXY-OXOLAN-2-YL]ME...

MacromoleculeName: [(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
type: ligand / ID: 3 / Number of copies: 2 / Formula: AR6
Molecular weightTheoretical: 559.316 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration1.2 mg/mL
BufferpH: 6
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: liquid-nitrogen-cooled liquid ethane was used as the cryogen..

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 57.32 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: NONE
Startup modelType of model: INSILICO MODEL / In silico model: AlphaFold model
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 152750
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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