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- EMDB-16233: Helical structure of BcThsA in complex with 1''-3'gcADPR -

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Basic information

Entry
Database: EMDB / ID: EMD-16233
TitleHelical structure of BcThsA in complex with 1''-3'gcADPR
Map dataSharpened EM map
Sample
  • Complex: BcThsA in complex with 1''-3'gcADPR
    • Protein or peptide: NAD(+) hydrolase ThsA
  • Ligand: (2R,3R,3aS,5S,6R,7S,8R,11R,13S,15aR)-2-(6-amino-9H-purin-9-yl)-3,6,7,11,13-pentahydroxyoctahydro-2H,5H,11H,13H-5,8-epoxy-11lambda~5~,13lambda~5~-furo[2,3-g][1,3,5,9,2,4]tetraoxadiphosphacyclotetradecine-11,13-dione
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE
KeywordsThoeris / SIR2 domain / SLOG domain / 3'cADPR / HYDROLASE
Function / homology
Function and homology information


NAD+ glycohydrolase / defense response to virus / hydrolase activity / nucleotide binding / cytoplasm
Similarity search - Function
NAD(+) hydrolase ThsA, Sir2/TIR-associating SLOG domain / Sir2- and TIR-associating SLOG family / SIR2-like domain / SIR2-like domain / Sirtuin family, catalytic core domain / Sirtuin catalytic domain profile. / DHS-like NAD/FAD-binding domain superfamily
Similarity search - Domain/homology
NAD(+) hydrolase ThsA
Similarity search - Component
Biological speciesBacillus cereus (bacteria) / Bacillus cereus MSX-D12 (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 2.96 Å
AuthorsTamulaitiene G / Sasnauskas G / Sabonis D
Funding supportLithuania, 1 items
OrganizationGrant numberCountry
Research Council of LithuaniaS-MIP-21-6Lithuania
CitationJournal: Nature / Year: 2024
Title: Activation of Thoeris antiviral system via SIR2 effector filament assembly.
Authors: Giedre Tamulaitiene / Dziugas Sabonis / Giedrius Sasnauskas / Audrone Ruksenaite / Arunas Silanskas / Carmel Avraham / Gal Ofir / Rotem Sorek / Mindaugas Zaremba / Virginijus Siksnys /
Abstract: To survive bacteriophage (phage) infections, bacteria developed numerous anti-phage defence systems. Some of them (for example, type III CRISPR-Cas, CBASS, Pycsar and Thoeris) consist of two modules: ...To survive bacteriophage (phage) infections, bacteria developed numerous anti-phage defence systems. Some of them (for example, type III CRISPR-Cas, CBASS, Pycsar and Thoeris) consist of two modules: a sensor responsible for infection recognition and an effector that stops viral replication by destroying key cellular components. In the Thoeris system, a Toll/interleukin-1 receptor (TIR)-domain protein, ThsB, acts as a sensor that synthesizes an isomer of cyclic ADP ribose, 1''-3' glycocyclic ADP ribose (gcADPR), which is bound in the Smf/DprA-LOG (SLOG) domain of the ThsA effector and activates the silent information regulator 2 (SIR2)-domain-mediated hydrolysis of a key cell metabolite, NAD (refs. ). Although the structure of ThsA has been solved, the ThsA activation mechanism remained incompletely understood. Here we show that 1''-3' gcADPR, synthesized in vitro by the dimeric ThsB' protein, binds to the ThsA SLOG domain, thereby activating ThsA by triggering helical filament assembly of ThsA tetramers. The cryogenic electron microscopy (cryo-EM) structure of activated ThsA revealed that filament assembly stabilizes the active conformation of the ThsA SIR2 domain, enabling rapid NAD depletion. Furthermore, we demonstrate that filament formation enables a switch-like response of ThsA to the 1''-3' gcADPR signal.
History
DepositionNov 29, 2022-
Header (metadata) releaseFeb 21, 2024-
Map releaseFeb 21, 2024-
UpdateApr 3, 2024-
Current statusApr 3, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16233.png

Downloads & links

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Map

FileDownload / File: emd_16233.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened EM map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 320 pix.
= 352. Å		1.1 Å/pix.
x 320 pix.
= 352. Å		1.1 Å/pix.
x 320 pix.
= 352. Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.78
Minimum - Maximum-5.823351 - 8.518079999999999
Average (Standard dev.)0.002784182 (±0.33646688)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 352.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16233_msk_1.map
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Additional map: Unsharpened EM map

Fileemd_16233_additional_1.map
AnnotationUnsharpened EM map
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Additional map: Local refinement (mask on A subunit) sharpened EM...

Fileemd_16233_additional_2.map
AnnotationLocal refinement (mask on A subunit) sharpened EM map resampled on main EM map
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Additional map: Local refinement (mask on A subunit) EM map resampled on main EM map

Fileemd_16233_additional_3.map
AnnotationLocal refinement (mask on A subunit) EM map resampled on main EM map
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Additional map: Local refinement (mask on A subunit) EM half...

Fileemd_16233_additional_4.map
AnnotationLocal refinement (mask on A subunit) EM half map resampled on main EM map
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Additional map: Local refinement (mask on A subunit) EM half...

Fileemd_16233_additional_5.map
AnnotationLocal refinement (mask on A subunit) EM half map resampled on main EM map
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Half map: #2

Fileemd_16233_half_map_1.map
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Half map: #1

Fileemd_16233_half_map_2.map
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Sample components

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Entire : BcThsA in complex with 1''-3'gcADPR

EntireName: BcThsA in complex with 1''-3'gcADPR
Components
  • Complex: BcThsA in complex with 1''-3'gcADPR
    • Protein or peptide: NAD(+) hydrolase ThsA
  • Ligand: (2R,3R,3aS,5S,6R,7S,8R,11R,13S,15aR)-2-(6-amino-9H-purin-9-yl)-3,6,7,11,13-pentahydroxyoctahydro-2H,5H,11H,13H-5,8-epoxy-11lambda~5~,13lambda~5~-furo[2,3-g][1,3,5,9,2,4]tetraoxadiphosphacyclotetradecine-11,13-dione
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

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Supramolecule #1: BcThsA in complex with 1''-3'gcADPR

SupramoleculeName: BcThsA in complex with 1''-3'gcADPR / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Bacillus cereus (bacteria)

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Macromolecule #1: NAD(+) hydrolase ThsA

MacromoleculeName: NAD(+) hydrolase ThsA / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO / EC number: NAD+ glycohydrolase
Source (natural)Organism: Bacillus cereus MSX-D12 (bacteria)
Molecular weightTheoretical: 55.417746 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SGGMKMNPIV ELFIKDFTKE VMEENAAIFA GAGLSMSVGY VSWAKLLEPI AQEIGLDVNK ENDLVSLAQY YCNENQGNRG RINQIILDE FSRKVDLTEN HKILARLPIH TYWTTAYDRL IEKALEEENK IADVKYTVKQ LATTKVKRDA VVYKMHGDVE H PSEAVLIK ...String:
SGGMKMNPIV ELFIKDFTKE VMEENAAIFA GAGLSMSVGY VSWAKLLEPI AQEIGLDVNK ENDLVSLAQY YCNENQGNRG RINQIILDE FSRKVDLTEN HKILARLPIH TYWTTAYDRL IEKALEEENK IADVKYTVKQ LATTKVKRDA VVYKMHGDVE H PSEAVLIK DDYEKYSIKM DPYIKALSGD LVSKTFLFVG FSFTDPNLDY ILSRVRSAYE RDQRRHYCLI KKEERRPDEL EA DFEYRVR KQELFISDLS RFNIKTIVLN NYNEITEILQ RIENNIKTKT VFLSGSAVEY NHWETEHAEQ FIHQLSKELI RKD FNIVSG FGLGVGSFVI NGVLEELYMN QGTIDDDRLI LRPFPQGKKG EEQWDKYRRD MITRTGVSIF LYGNKIDKGQ VVKA KGVQS EFNISFEQNN YVVPVGATGY IAKDLWNKVN EEFETYYPGA DARMKKLFGE LNNEALSIEE LINTIIEFVE ILSN

UniProtKB: NAD(+) hydrolase ThsA

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Macromolecule #2: (2R,3R,3aS,5S,6R,7S,8R,11R,13S,15aR)-2-(6-amino-9H-purin-9-yl)-3,...

MacromoleculeName: (2R,3R,3aS,5S,6R,7S,8R,11R,13S,15aR)-2-(6-amino-9H-purin-9-yl)-3,6,7,11,13-pentahydroxyoctahydro-2H,5H,11H,13H-5,8-epoxy-11lambda~5~,13lambda~5~-furo[2,3-g][1,3,5,9,2,4]tetraoxadiphosphacyclotetradecine-11,13-dione
type: ligand / ID: 2 / Number of copies: 12 / Formula: OJC
Molecular weightTheoretical: 541.3 Da
Chemical component information

ChemComp-OJC:
(2R,3R,3aS,5S,6R,7S,8R,11R,13S,15aR)-2-(6-amino-9H-purin-9-yl)-3,6,7,11,13-pentahydroxyoctahydro-2H,5H,11H,13H-5,8-epoxy-11lambda~5~,13lambda~5~-furo[2,3-g][1,3,5,9,2,4]tetraoxadiphosphacyclotetradecine-11,13-dione

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Macromolecule #3: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

MacromoleculeName: NICOTINAMIDE-ADENINE-DINUCLEOTIDE / type: ligand / ID: 3 / Number of copies: 12 / Formula: NAD
Molecular weightTheoretical: 663.425 Da
Chemical component information

ChemComp-NAD:
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / NAD*YM / Nicotinamide adenine dinucleotide

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration1.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
20.0 mMNa-Hepes
150.0 mMsodium chloride
1.0 mMDTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 2321 / Average exposure time: 46.33 sec. / Average electron dose: 30.64 e/Å2

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6lhx

Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Helical parameters - Δz: 41.87 Å
Applied symmetry - Helical parameters - Δ&Phi: 128.947 °
Applied symmetry - Helical parameters - Axial symmetry: D2 (2x2 fold dihedral)
Resolution.type: BY AUTHOR / Resolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 233008
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8bto:
Helical structure of BcThsA in complex with 1''-3'gcADPR

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