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- EMDB-45466: CryoEM structure of CRISPR associated effector, CARF-Adenosine de... -

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Basic information

Entry
Database: EMDB / ID: EMD-45466
TitleCryoEM structure of CRISPR associated effector, CARF-Adenosine deaminase 1, Cad1, in cA6 (partial density) bound form with ATP (partial density).
Map datastructure of CRISPR associated effector, CARF-Adenosine deaminase 1, Cad1, in cA6 (partial density) bound form with ATP (partial density).
Sample
  • Complex: cA6 (partial density)-Cad1-ATP (partial density)
    • Protein or peptide: Adenosine deaminase domain-containing protein
    • RNA: RNA (5'-R(P*AP*AP*AP*AP*AP*A)-3')
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsAntiphage defense / CRISPR / Deamination / CARF-Adenosine deaminase / ATP / ANTIVIRAL PROTEIN / 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
Adenosine deaminase domain-containing protein
Similarity search - Component
Biological speciesbacteroidale bacteria (bacteria) / Bacteroidales bacterium (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsMajumder P / Patel DJ
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIHGM 129430, NIHGM 145888 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 24, 2024-
Header (metadata) releaseOct 30, 2024-
Map releaseOct 30, 2024-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45466.png

Downloads & links

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Map

FileDownload / File: emd_45466.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationstructure of CRISPR associated effector, CARF-Adenosine deaminase 1, Cad1, in cA6 (partial density) bound form with ATP (partial density).
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.81 Å/pix.
x 512 pix.
= 414.208 Å		0.81 Å/pix.
x 512 pix.
= 414.208 Å		0.81 Å/pix.
x 512 pix.
= 414.208 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.809 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.128
Minimum - Maximum-0.18310146 - 0.61951023
Average (Standard dev.)0.0015684272 (±0.020588228)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 414.208 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map A

Fileemd_45466_half_map_1.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B

Fileemd_45466_half_map_2.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : cA6 (partial density)-Cad1-ATP (partial density)

EntireName: cA6 (partial density)-Cad1-ATP (partial density)
Components
  • Complex: cA6 (partial density)-Cad1-ATP (partial density)
    • Protein or peptide: Adenosine deaminase domain-containing protein
    • RNA: RNA (5'-R(P*AP*AP*AP*AP*AP*A)-3')
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: cA6 (partial density)-Cad1-ATP (partial density)

SupramoleculeName: cA6 (partial density)-Cad1-ATP (partial density) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Cad1 forms a trimer of dimers. In this structure partial density of bound cA6 is visible. ATP is present.
Source (natural)Organism: bacteroidale bacteria (bacteria)
Molecular weightTheoretical: 400 KDa

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Macromolecule #1: Adenosine deaminase domain-containing protein

MacromoleculeName: Adenosine deaminase domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Bacteroidales bacterium (bacteria)
Molecular weightTheoretical: 67.264945 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSRVLLCSAG HSSMVVPEAF HAVPEGFEEV HVFTTDSEKF NPVVLNDFFH SLPNVRFSIT KCHGLADILN ERDFEFYQEM LWQWYLTKM PDNELPYVCL SGGIKSMSAS LQKAATLFGA QSVFHVLADN NPRNIEEMFD ALQKGQIHFI EMGYEPGWAA L RRLKKILP ...String:
MSRVLLCSAG HSSMVVPEAF HAVPEGFEEV HVFTTDSEKF NPVVLNDFFH SLPNVRFSIT KCHGLADILN ERDFEFYQEM LWQWYLTKM PDNELPYVCL SGGIKSMSAS LQKAATLFGA QSVFHVLADN NPRNIEEMFD ALQKGQIHFI EMGYEPGWAA L RRLKKILP INEGCSRDNF KPLISKSIEE ILSNVKIMAS DTGKSNQLPF PSLAILPPIA QQWLQLPLSA NDGAWIQNLP KV DLHCHLG GFATSGSLLD QVRGAASEPD LIDRTFSPQE IAGWPRSHKS ISLDKYMELG NANGSKLLKD KGCLIRQVEL LYQ SLVNDN VAYAEIRCSP NNYADKNKNR SAWVVLQDIN DTFTRLITEA KQKNQFYCHV NLLVIASRKF SGDLSDISKH LALA ITAMQ QGEGVCRIVG VDLAGFENKE TRASYYEHDF KAVHRCGLAV TAHAGENDDP EGIWQAVYSL HARRLGHALN LLEAP DLMR TVIERKIGVE MCPYANYQIK GFAPMPNFSA LYPLKKYLEA GILVSVNTDN IGISGANLSE NLLILADLCP GISRMD VLT IIRNSIETAF ISHDFRMELL KFFDRKIYDV CLISIKN

UniProtKB: Adenosine deaminase domain-containing protein

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

MacromoleculeName: RNA (5'-R(P*AP*AP*AP*AP*AP*A)-3') / type: rna / ID: 2 / Number of copies: 3
Source (natural)Organism: Bacteroidales bacterium (bacteria)
Molecular weightTheoretical: 1.930277 KDa
SequenceString:
AAAAAA

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 12 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.7 mg/mL
BufferpH: 8
Details: 25 mM Hepes pH 8, 500 mM NaCl, 2 mM Beta-Mercaptoethanol and 5 percentage glycerol
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Blot time 2.5 s, Wait time 12s, Blot force 0..
DetailsCad1 protein mixed with cA6 and ATP. Incubated overnight at 310.15 K.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 61.0 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: 2.4 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

Startup modelType of model: INSILICO MODEL / In silico model: AlphaFold2 model
Final reconstructionNumber classes used: 2 / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 271802
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementProtocol: AB INITIO MODEL
Output model

PDB-9cdb:
CryoEM structure of CRISPR associated effector, CARF-Adenosine deaminase 1, Cad1, in cA6 (partial density) bound form with ATP (partial density).

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