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- EMDB-18486: CryoEM structure of the apo SPARTA (BabAgo/TIR-APAZ) complex -

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Basic information

Entry
Database: EMDB / ID: EMD-18486
TitleCryoEM structure of the apo SPARTA (BabAgo/TIR-APAZ) complex
Map data
Sample
  • Complex: Complex of BabAgo with BabTIR-APAZ
    • Protein or peptide: Short prokaryotic Argonaute
    • Protein or peptide: Toll/interleukin-1 receptor domain-containing protein
KeywordsProkaryotic Argonaute / TIR domain / RNA binding protein / DNA binding protein / IMMUNE SYSTEM
Biological speciesBacillales bacterium (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.57 Å
AuthorsFinocchio G / Koopal B / Potocnik A / Heijstek C / Jinek M / Swarts D
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)European Union
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Target DNA-dependent activation mechanism of the prokaryotic immune system SPARTA.
Authors: Giada Finocchio / Balwina Koopal / Ana Potocnik / Clint Heijstek / Adrie H Westphal / Martin Jinek / Daan C Swarts /
Abstract: In both prokaryotic and eukaryotic innate immune systems, TIR domains function as NADases that degrade the key metabolite NAD+ or generate signaling molecules. Catalytic activation of TIR domains ...In both prokaryotic and eukaryotic innate immune systems, TIR domains function as NADases that degrade the key metabolite NAD+ or generate signaling molecules. Catalytic activation of TIR domains requires oligomerization, but how this is achieved varies in distinct immune systems. In the Short prokaryotic Argonaute (pAgo)/TIR-APAZ (SPARTA) immune system, TIR NADase activity is triggered upon guide RNA-mediated recognition of invading DNA by an unknown mechanism. Here, we describe cryo-EM structures of SPARTA in the inactive monomeric and target DNA-activated tetrameric states. The monomeric SPARTA structure reveals that in the absence of target DNA, a C-terminal tail of TIR-APAZ occupies the nucleic acid binding cleft formed by the pAgo and TIR-APAZ subunits, inhibiting SPARTA activation. In the active tetrameric SPARTA complex, guide RNA-mediated target DNA binding displaces the C-terminal tail and induces conformational changes in pAgo that facilitate SPARTA-SPARTA dimerization. Concurrent release and rotation of one TIR domain allow it to form a composite NADase catalytic site with the other TIR domain within the dimer, and generate a self-complementary interface that mediates cooperative tetramerization. Combined, this study provides critical insights into the structural architecture of SPARTA and the molecular mechanism underlying target DNA-dependent oligomerization and catalytic activation.
History
DepositionSep 20, 2023-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18486.png

Downloads & links

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Map

FileDownload / File: emd_18486.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.65 Å
Density
Contour LevelBy AUTHOR: 0.0653
Minimum - Maximum-0.21047755 - 0.34800074
Average (Standard dev.)-0.00008986277 (±0.008131958)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 233.99998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_18486_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_18486_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of BabAgo with BabTIR-APAZ

EntireName: Complex of BabAgo with BabTIR-APAZ
Components
  • Complex: Complex of BabAgo with BabTIR-APAZ
    • Protein or peptide: Short prokaryotic Argonaute
    • Protein or peptide: Toll/interleukin-1 receptor domain-containing protein

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Supramolecule #1: Complex of BabAgo with BabTIR-APAZ

SupramoleculeName: Complex of BabAgo with BabTIR-APAZ / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bacillales bacterium (bacteria)

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Macromolecule #1: Short prokaryotic Argonaute

MacromoleculeName: Short prokaryotic Argonaute / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bacillales bacterium (bacteria)
Molecular weightTheoretical: 57.911199 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKELIYIHEP NILFANGQKC ADPRDGLALF GPFTKIYGIK SGVVGTQYGL SIFKNYINHI QKPIYNANNI TRPMFPGFEA VFGCKWDAD NVVFKEVTKE EIEKILYTES NHKRTYDLVS LFINKIITAN KNEDEKVDVW FLVIPDEIYQ YCRPNSVLPK D LVQTKSLI ...String:
MKELIYIHEP NILFANGQKC ADPRDGLALF GPFTKIYGIK SGVVGTQYGL SIFKNYINHI QKPIYNANNI TRPMFPGFEA VFGCKWDAD NVVFKEVTKE EIEKILYTES NHKRTYDLVS LFINKIITAN KNEDEKVDVW FLVIPDEIYQ YCRPNSVLPK D LVQTKSLI TKSKAKSFRY EPTLFENINK ELKEQEKEAI TYNYDAQFHD QLKARLLEHT IPTQILREST LAWRDFKNKF GA PKRDFSK IEGHLAWTIS TAAFYKAGGK PWKLSDIRSG VCYLGLVYKQ IEKSSNPKNA CCAAQMFLDN GDGTVFKGEV GPW YNQEKH EFHLNPKEAK ALLTQALNSY KEQNGVFPKE IFIHAKTKFN GQEWNAFQEV TPEGTNLVGV TITKTKPLKL FKSE GNYPI MRGNAFIVNE RSAFLWTVGY VPKTESTLSM EVPNPIFIEI NKGEADIEQV LKDVLALTKL NYNACIYADG VPVTL RFAD KIGEILTAST ELKAPPLAFK YYI

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Macromolecule #2: Toll/interleukin-1 receptor domain-containing protein

MacromoleculeName: Toll/interleukin-1 receptor domain-containing protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bacillales bacterium (bacteria)
Molecular weightTheoretical: 53.052355 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SNARNKIFIS HAAPDDNDFT KWLALKLIAL GYEVWCDVLF LDKGADFWKV IDKEIREGAI KFLLATSEIA IKRDGVLKEI AVAEKVKKQ LKDDNFIIPL IIDENLSYDD LPPEIIRLNA VDFKKSWAVG LQDLLKALDD QKVEKNSPDP DKSNALYQQI F LHNKGIIE ...String:
SNARNKIFIS HAAPDDNDFT KWLALKLIAL GYEVWCDVLF LDKGADFWKV IDKEIREGAI KFLLATSEIA IKRDGVLKEI AVAEKVKKQ LKDDNFIIPL IIDENLSYDD LPPEIIRLNA VDFKKSWAVG LQDLLKALDD QKVEKNSPDP DKSNALYQQI F LHNKGIIE REEIYDSNWF SILSFPKELR FHDYEKLMPK GFDVRELTYP AVRYKNYLCT FAWEYDFMHQ LPKTETYNSS QT IRIPTEE ILSGKYDSPF IGNFECQRLI VQLLNKAFEL RMKEKGVREY PMSNKMGYWF EKGKLEKDKF NKVLLVGKQK DKH WHFGIS AAGKLYPFPV LMISSHIFFT KDGKELIESK KIQHAARRRQ GKNWWNDDWR NKLLAFVKYL SDDENSFYLE VGSE EKIYI SNEPVQFVGK VSYNMPEKNN LKDEAEISDL NDLNEFDGEI FEETDSE

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 58.005 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: Model generated with AlphaFold2 ColabFold
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.57 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 904721

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