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- PDB-8btp: Helical structure of BcThsA in complex with 1''-3'gc(etheno)ADPR -

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Basic information

Entry
Database: PDB / ID: 8btp
TitleHelical structure of BcThsA in complex with 1''-3'gc(etheno)ADPR
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 / Sirtuin family, catalytic core domain / Sirtuin catalytic domain profile. / DHS-like NAD/FAD-binding domain superfamily
Similarity search - Domain/homology
ETHENO-NAD / : / NAD(+) hydrolase ThsA
Similarity search - Component
Biological speciesBacillus cereus MSX-D12 (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.75 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)680,04636
Polymers665,01312
Non-polymers15,03324
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization, DLS
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "D"
d_5ens_1chain "E"
d_6ens_1chain "F"
d_7ens_1chain "G"
d_8ens_1chain "H"
d_9ens_1chain "I"
d_10ens_1chain "J"
d_11ens_1chain "K"
d_12ens_1chain "L"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1LYSASNA1 - 473
d_12ens_1LTELTEB
d_13ens_1ENAENAC
d_21ens_1LYSASND1 - 473
d_22ens_1LTELTEE
d_23ens_1ENAENAF
d_31ens_1LYSASNG1 - 473
d_32ens_1LTELTEH
d_33ens_1ENAENAI
d_41ens_1LYSASNJ1 - 473
d_42ens_1LTELTEK
d_43ens_1ENAENAL
d_51ens_1LYSASNM1 - 473
d_52ens_1LTELTEN
d_53ens_1ENAENAO
d_61ens_1LYSASNP1 - 473
d_62ens_1LTELTEQ
d_63ens_1ENAENAR
d_71ens_1LYSASNS1 - 473
d_72ens_1LTELTET
d_73ens_1ENAENAU
d_81ens_1LYSASNV1 - 473
d_82ens_1LTELTEW
d_83ens_1ENAENAX
d_91ens_1LYSASNY1 - 473
d_92ens_1LTELTEZ
d_93ens_1ENAENAAA
d_101ens_1LYSASNJ1 - 473
d_102ens_1LTELTEJ
d_103ens_1ENAENAJ
d_111ens_1LYSASNK1 - 473
d_112ens_1LTELTEK
d_113ens_1ENAENAK
d_121ens_1LYSASNL1 - 473
d_122ens_1LTELTEL
d_123ens_1ENAENAL

NCS oper:
IDCodeMatrixVector
1given(0.628623007907, 0.77771015179, -0.000183667084111), (0.777710162842, -0.628622942723, 0.000313838289487), (0.000128617880859, -0.000340125727426, -0.999999933886)-71.4528586256, 149.721467158, 310.133074159
2given(-0.628560181054, -0.777760947946, 8.15074822465E-5), (-0.777760918735, 0.628560119793, -0.000359297965136), (0.000228215573154, -0.000289233728294, -0.999999932131)423.469612986, 202.304870676, 310.105837928
3given(-0.999999987337, 0.000128142796422, -9.43692904785E-5), (-0.000128163306591, -0.999999968163, 0.000217365472087), (-9.43414336547E-5, 0.000217377564015, 0.999999971923)351.973845623, 351.980705864, -0.0222818075761
4given(0.628721898282, -0.77763019303, 0.000239811076705), (0.777630220479, 0.628721908958, -3.73428777788E-5), (-0.00012173552868, 0.000209962625456, 0.999999970548)202.168321486, -71.502363907, -41.9103529397
5given(-0.209747754886, 0.97775538866, -0.000528457272272), (0.977755507728, 0.209747838265, 0.000107009492787), (0.000215471878684, -0.000494257007699, -0.999999854641)40.9335879305, -32.9786241858, 268.244807352
6given(0.209863211607, -0.977730673489, 0.000403153217687), (-0.977730708946, -0.209863312283, -0.000225703793011), (0.000305284591171, -0.00034680835847, -0.999999893263)311.068277704, 385.029454757, 268.202094642
7given(-0.62856097884, 0.777760274508, -0.000226443920097), (-0.777760301526, -0.628560983397, 5.9342592779E-5), (-9.61795018509E-5, 0.000213419529778, 0.999999972601)149.776345092, 423.490808303, -41.9166815095
8given(0.628839765682, 0.777534906118, -0.000137354114368), (-0.77753491098, 0.628839725833, -0.000247836226851), (-0.000106327593756, 0.000262646893908, 0.999999959856)-71.4901854835, 202.210743602, 41.8822709826
9given(0.999999850534, 0.000482274126395, 0.00025757210305), (0.000482155926599, -0.99999977853, 0.000458764948557), (0.00025779329647, -0.000458640690072, -0.999999861596)-0.0897504969827, 351.816937345, 352.035049534
10given(-0.99999981704, -0.000565235056109, -0.000215476708731), (-0.000565151744447, 0.999999765609, -0.000386503847296), (0.000215695123749, -0.000386381999543, -0.999999902092)352.114786589, 0.179388276475, 352.029817896
11given(-0.629006009946, -0.777400436679, 2.24129434763E-5), (0.777400424312, -0.629005994207, 0.000198825868456), (-0.000140469441167, 0.00014248649796, 0.999999979983)423.513720655, 149.857631251, 41.9064528222

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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-RK3 / (1S,3S,4R,5R,7R,15R,16S,17R)-5-imidazo[2,1-f]purin-3-yl-10,12-bis(oxidanyl)-10,12-bis(oxidanylidene)-2,6,9,11,13,18-hexaoxa-10$l^{5},12$l^{5}-diphosphatricyclo[13.2.1.0^{3,7}]octadecane-4,16,17-triol / 1''-3'gc(etheno)ADPR


Mass: 565.322 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C17H21N5O13P2
#3: Chemical
ChemComp-ENA / ETHENO-NAD


Type: RNA linking / Mass: 687.447 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C23H27N7O14P2 / Feature type: SUBJECT OF INVESTIGATION
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'gc(etheno)ADPR / 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 FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 30.33 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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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: 41.935 Å / Axial symmetry: D2
3D reconstructionResolution: 2.75 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 935056 / 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: 20.67 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005547232
ELECTRON MICROSCOPYf_angle_d0.677664032
ELECTRON MICROSCOPYf_chiral_restr0.51617188
ELECTRON MICROSCOPYf_plane_restr0.0038064
ELECTRON MICROSCOPYf_dihedral_angle_d12.369518816
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints0.0756437980977
ens_1d_3AELECTRON MICROSCOPYNCS constraints0.0300985726535
ens_1d_4AELECTRON MICROSCOPYNCS constraints0.0773464305517
ens_1d_5AELECTRON MICROSCOPYNCS constraints0.0508220967925
ens_1d_6AELECTRON MICROSCOPYNCS constraints0.0617187905591
ens_1d_7AELECTRON MICROSCOPYNCS constraints0.0690579450686
ens_1d_8AELECTRON MICROSCOPYNCS constraints0.0489481546365
ens_1d_9AELECTRON MICROSCOPYNCS constraints0.0837112294437
ens_1d_10AELECTRON MICROSCOPYNCS constraints0.0376148894159
ens_1d_11AELECTRON MICROSCOPYNCS constraints0.081311398463
ens_1d_12AELECTRON MICROSCOPYNCS constraints0.0576329813554

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