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- EMDB-41966: Cryo-EM structure of the SPARTA oligomer with guide RNA and target DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-41966
TitleCryo-EM structure of the SPARTA oligomer with guide RNA and target DNA
Map datacomposite map
Sample
  • Complex: SPARTA complex with gRNA/tDNA
    • RNA: RNA (5'-R(P*UP*GP*AP*GP*GP*UP*AP*GP*UP*AP*GP*GP*UP*UP*GP*UP*AP*UP*A)-3')
    • DNA: DNA (5'-D(P*TP*AP*TP*AP*CP*AP*AP*CP*CP*TP*AP*CP*TP*AP*CP*CP*TP*C)-3')
    • Protein or peptide: TIR domain-containing protein
    • Protein or peptide: Piwi domain-containing protein
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsRNA / DNA / Argonaute / TIR / immune system / RNA BINDING PROTEIN-RNA-DNA complex
Function / homologyTIR domain / Toll/interleukin-1 receptor homology (TIR) domain / Toll/interleukin-1 receptor homology (TIR) domain superfamily / nucleic acid binding / Ribonuclease H superfamily / Ribonuclease H-like superfamily / signal transduction / Piwi domain-containing protein / TIR domain-containing protein
Function and homology information
Biological speciesThermoflavifilum thermophilum (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsMalik R / Kottur J / Aggarwal AK
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI) United States
CitationJournal: Nat Commun / Year: 2024
Title: Nucleic acid mediated activation of a short prokaryotic Argonaute immune system.
Authors: Jithesh Kottur / Radhika Malik / Aneel K Aggarwal /
Abstract: A short prokaryotic Argonaute (pAgo) TIR-APAZ (SPARTA) defense system, activated by invading DNA to unleash its TIR domain for NAD(P) hydrolysis, was recently identified in bacteria. We report the ...A short prokaryotic Argonaute (pAgo) TIR-APAZ (SPARTA) defense system, activated by invading DNA to unleash its TIR domain for NAD(P) hydrolysis, was recently identified in bacteria. We report the crystal structure of SPARTA heterodimer in the absence of guide-RNA/target-ssDNA (2.66 Å) and a cryo-EM structure of the SPARTA oligomer (tetramer of heterodimers) bound to guide-RNA/target-ssDNA at nominal 3.15-3.35 Å resolution. The crystal structure provides a high-resolution view of SPARTA, revealing the APAZ domain as equivalent to the N, L1, and L2 regions of long pAgos and the MID domain containing a unique insertion (insert57). Cryo-EM structure reveals regions of the PIWI (loop10-9) and APAZ (helix αN) domains that reconfigure for nucleic-acid binding and decrypts regions/residues that reorganize to expose a positively charged pocket for higher-order assembly. The TIR domains amass in a parallel-strands arrangement for catalysis. We visualize SPARTA before and after RNA/ssDNA binding and uncover the basis of its active assembly leading to abortive infection.
History
DepositionSep 14, 2023-
Header (metadata) releaseJun 12, 2024-
Map releaseJun 12, 2024-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41966.png

Downloads & links

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Map

FileDownload / File: emd_41966.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcomposite map
Voxel sizeX=Y=Z: 1.083 Å
Density
Contour LevelBy AUTHOR: 0.564
Minimum - Maximum-0.4118225 - 4.538744
Average (Standard dev.)0.0246301 (±0.06510961)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 519.83997 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : SPARTA complex with gRNA/tDNA

EntireName: SPARTA complex with gRNA/tDNA
Components
  • Complex: SPARTA complex with gRNA/tDNA
    • RNA: RNA (5'-R(P*UP*GP*AP*GP*GP*UP*AP*GP*UP*AP*GP*GP*UP*UP*GP*UP*AP*UP*A)-3')
    • DNA: DNA (5'-D(P*TP*AP*TP*AP*CP*AP*AP*CP*CP*TP*AP*CP*TP*AP*CP*CP*TP*C)-3')
    • Protein or peptide: TIR domain-containing protein
    • Protein or peptide: Piwi domain-containing protein
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: SPARTA complex with gRNA/tDNA

SupramoleculeName: SPARTA complex with gRNA/tDNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Thermoflavifilum thermophilum (bacteria)

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Macromolecule #1: RNA (5'-R(P*UP*GP*AP*GP*GP*UP*AP*GP*UP*AP*GP*GP*UP*UP*GP*UP*AP*UP...

MacromoleculeName: RNA (5'-R(P*UP*GP*AP*GP*GP*UP*AP*GP*UP*AP*GP*GP*UP*UP*GP*UP*AP*UP*A)-3')
type: rna / ID: 1 / Number of copies: 4
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.812045 KDa
SequenceString:
UGAGGUAGUA GGUUGUAUAG U

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Macromolecule #2: DNA (5'-D(P*TP*AP*TP*AP*CP*AP*AP*CP*CP*TP*AP*CP*TP*AP*CP*CP*TP*C)-3')

MacromoleculeName: DNA (5'-D(P*TP*AP*TP*AP*CP*AP*AP*CP*CP*TP*AP*CP*TP*AP*CP*CP*TP*C)-3')
type: dna / ID: 2 / Number of copies: 4 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.703841 KDa
SequenceString:
(DA)(DA)(DA)(DC)(DG)(DA)(DC)(DG)(DG)(DC) (DC)(DA)(DG)(DT)(DG)(DC)(DC)(DA)(DA)(DG) (DC)(DT)(DT)(DA)(DC)(DT)(DA)(DT)(DA) (DC)(DA)(DA)(DC)(DC)(DT)(DA)(DC)(DT)(DA) (DC) (DC)(DT)(DC)(DA)(DT)

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Macromolecule #3: TIR domain-containing protein

MacromoleculeName: TIR domain-containing protein / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Thermoflavifilum thermophilum (bacteria)
Molecular weightTheoretical: 53.256734 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRNKIFISHA TPEDDDFTRW LSLKLIGLGY EVWCDILFLD KGVDFWSTIE KEIRENTCKF LIVSSTAGNK REGVLKELAV ATKVKKHLQ DDMFIIPLAI DENLSYDDIN IEIVRLNAID FKKSWAKGLQ DLLDAFEKQN VPKKPPDHSK SNLLYQQIFL H DKQAIEKE ...String:
MRNKIFISHA TPEDDDFTRW LSLKLIGLGY EVWCDILFLD KGVDFWSTIE KEIRENTCKF LIVSSTAGNK REGVLKELAV ATKVKKHLQ DDMFIIPLAI DENLSYDDIN IEIVRLNAID FKKSWAKGLQ DLLDAFEKQN VPKKPPDHSK SNLLYQQIFL H DKQAIEKE ETYDSNWFPI ISFPNELRFH RYDWRLPKQF DVRTLAFPAI RYKEYLCTFA WEYDFIHQLP KTETYNGQES IR ISTSDIL SGRYDTDFIR NYECQRLIVQ LINKAFELRM KDKNVREYQM SKTFAYWIEK GKLEKDKFEK IKLVGKQKNK YWH FGISAA GKLYPSPVLM VSSHIIFTMD GINLIKSKSI QHSSRRKQGK NWWNDKWREK LLAFIRFLSD DQNAIYLNVG SEEK ILISN KPLKFFGKMS YVTPSEVTLE EESVLADINN FEEDTEDLDE LEDIE

UniProtKB: TIR domain-containing protein

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Macromolecule #4: Piwi domain-containing protein

MacromoleculeName: Piwi domain-containing protein / type: protein_or_peptide / ID: 4 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Thermoflavifilum thermophilum (bacteria)
Molecular weightTheoretical: 58.304848 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKELIYIEEP SILFAHGQKC TDPRDGLALF GPLNQIYGIK SGVVGTQKGL QIFKSYLDKI QKPIYNHNNI TRPMFPGFEA VFGCKWESQ NIVFKEITDE EIRRYLFNAS THKRTYDLVT LFNDKIITAN KNDEERVDVW FVIVPEEIYK YCRPNSVLPN E LVQTKSLI ...String:
MKELIYIEEP SILFAHGQKC TDPRDGLALF GPLNQIYGIK SGVVGTQKGL QIFKSYLDKI QKPIYNHNNI TRPMFPGFEA VFGCKWESQ NIVFKEITDE EIRRYLFNAS THKRTYDLVT LFNDKIITAN KNDEERVDVW FVIVPEEIYK YCRPNSVLPN E LVQTKSLI SKSKAKSFRY TPTLFEEFNK KLKEVEKEAK TYNYDAQFHD QLKARLLEHT IPTQILREST LAWRDFKNTF GA PIRDFSK IEGHLAWTIS TAAYYKAGGK PWKLGDIRPG VCYLGLVYKK IEKSKNPQNA CCAAQMFLDN GDGTVFKGEV GPW YNPEKG EYHLKPKEAK ALLTQALESY KEQNKSYPKE VFIHARTRFN DEEWNAFNEV TPKNTNLVGV TITKSKPLKL YKTE GAFPI MRGNAYIVDE KKAFLWTLGF VPKLQSTLSM EVPNPIFIEI NKGEAEIQQV LKDILALTKL NYNACIYADG EPVTL RFAN KIGEILTAST EIKTPPLAFK YYI

UniProtKB: Piwi domain-containing protein

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 57 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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
Sugar embeddingMaterial: Vitreous ice
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 51.11 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Details: Composite cryo-EM map / Number images used: 238432
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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