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

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Basic information

Entry
Database: PDB / ID: 8bto
TitleHelical structure of BcThsA in complex with 1''-3'gcADPR
ComponentsNAD(+) hydrolase ThsA
KeywordsHYDROLASE / Thoeris / SIR2 domain / SLOG domain / 3'cADPR
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
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / Chem-OJC / NAD(+) hydrolase ThsA
Similarity search - Component
Biological speciesBacillus cereus MSX-D12 (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.96 Å
AuthorsTamulaitiene, G. / Sasnauskas, G. / Sabonis, D.
Funding supportLithuania, 1items
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, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 21, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Mar 27, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Apr 3, 2024Group: Database references / Category: citation_author / Item: _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: NAD(+) hydrolase ThsA
B: NAD(+) hydrolase ThsA
C: NAD(+) hydrolase ThsA
D: NAD(+) hydrolase ThsA
E: NAD(+) hydrolase ThsA
F: NAD(+) hydrolase ThsA
G: NAD(+) hydrolase ThsA
H: NAD(+) hydrolase ThsA
I: NAD(+) hydrolase ThsA
J: NAD(+) hydrolase ThsA
K: NAD(+) hydrolase ThsA
L: NAD(+) hydrolase ThsA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)679,47036
Polymers665,01312
Non-polymers14,45724
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering, DLS
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area70000 Å2
ΔGint-76 kcal/mol
Surface area209260 Å2

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Components

#1: Protein
NAD(+) hydrolase ThsA / NADase ThsA / Thoeris protein ThsA


Mass: 55417.746 Da / Num. of mol.: 12 / Mutation: N112A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus cereus MSX-D12 (bacteria) / Gene: thsA, II9_05448 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: J8G6Z1, NAD+ glycohydrolase
#2: Chemical
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


Mass: 541.300 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C15H21N5O13P2 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-NAD / NICOTINAMIDE-ADENINE-DINUCLEOTIDE / Nicotinamide adenine dinucleotide


Mass: 663.425 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C21H27N7O14P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: NAD*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: BcThsA in complex with 1''-3'gcADPR / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Bacillus cereus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
120 mMNa-Hepes1
2150 mMsodium chloride1
31 mMDTT1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 46.33 sec. / Electron dose: 30.64 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2321

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameCategory
2EPUimage acquisition
7UCSF ChimeraXmodel fitting
9PHENIXmodel refinement
13cryoSPARC3D reconstruction
CTF correctionType: NONE
Helical symmertyAngular rotation/subunit: 128.947 ° / Axial rise/subunit: 41.87 Å / Axial symmetry: D2
3D reconstructionResolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 233008 / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 56.29 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.007247076
ELECTRON MICROSCOPYf_angle_d0.696263828
ELECTRON MICROSCOPYf_chiral_restr0.04537152
ELECTRON MICROSCOPYf_plane_restr0.00388076
ELECTRON MICROSCOPYf_dihedral_angle_d12.271918012

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