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- EMDB-35419: Cryo-EM structure of monomeric SPARTA gRNA-ssDNA target complex -

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Basic information

Entry
Database: EMDB / ID: EMD-35419
TitleCryo-EM structure of monomeric SPARTA gRNA-ssDNA target complex
Map data
Sample
  • Complex: CrtSPARTA-gRNA-tDNA ternary complex
    • Protein or peptide: TIR domain-containing protein
    • Protein or peptide: Piwi domain-containing protein
    • RNA: guide RNA
    • DNA: target ssDNA
  • Ligand: MAGNESIUM ION
KeywordsRNA BINDING PROTEIN-RNA-DNA COMPLEX
Function / homologyTIR domain / Toll/interleukin-1 receptor homology (TIR) domain / Toll/interleukin-1 receptor homology (TIR) domain superfamily / Ribonuclease H superfamily / nucleic acid binding / Ribonuclease H-like superfamily / signal transduction / Piwi domain-containing protein / TIR domain-containing protein
Function and homology information
Biological speciesThermoflavifilum thermophilum (bacteria) / Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.54 Å
AuthorsZhang JT / Jia N / Huang HD
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Chem Biol / Year: 2024
Title: Target ssDNA activates the NADase activity of prokaryotic SPARTA immune system.
Authors: Jun-Tao Zhang / Xin-Yang Wei / Ning Cui / Ruilin Tian / Ning Jia /
Abstract: Argonaute proteins (Agos), which use small RNAs or DNAs as guides to recognize complementary nucleic acid targets, mediate RNA silencing in eukaryotes. In prokaryotes, Agos are involved in immunity: ...Argonaute proteins (Agos), which use small RNAs or DNAs as guides to recognize complementary nucleic acid targets, mediate RNA silencing in eukaryotes. In prokaryotes, Agos are involved in immunity: the short prokaryotic Ago/TIR-APAZ (SPARTA) immune system triggers cell death by degrading NAD in response to invading plasmids, but its molecular mechanisms remain unknown. Here we used cryo-electron microscopy to determine the structures of inactive monomeric and active tetrameric Crenotalea thermophila SPARTA complexes, revealing mechanisms underlying SPARTA assembly, RNA-guided recognition of target single-stranded DNA (ssDNA) and subsequent SPARTA tetramerization, as well as tetramerization-dependent NADase activation. The small RNA guides Ago to recognize its ssDNA target, inducing SPARTA tetramerization via both Ago- and TIR-mediated interactions and resulting in a two-stranded, parallel, head-to-tail TIR rearrangement primed for NAD hydrolysis. Our findings thus identify the molecular basis for target ssDNA-mediated SPARTA activation, which will facilitate the development of SPARTA-based biotechnological tools.
History
DepositionFeb 18, 2023-
Header (metadata) releaseJan 17, 2024-
Map releaseJan 17, 2024-
UpdateApr 10, 2024-
Current statusApr 10, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35419.png

Downloads & links

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Map

FileDownload / File: emd_35419.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum-8.320584999999999 - 11.465230999999999
Average (Standard dev.)-0.00014337219 (±0.2274443)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 281.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_35419_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Half map: #2

Fileemd_35419_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_35419_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : CrtSPARTA-gRNA-tDNA ternary complex

EntireName: CrtSPARTA-gRNA-tDNA ternary complex
Components
  • Complex: CrtSPARTA-gRNA-tDNA ternary complex
    • Protein or peptide: TIR domain-containing protein
    • Protein or peptide: Piwi domain-containing protein
    • RNA: guide RNA
    • DNA: target ssDNA
  • Ligand: MAGNESIUM ION

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Supramolecule #1: CrtSPARTA-gRNA-tDNA ternary complex

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

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Macromolecule #1: TIR domain-containing protein

MacromoleculeName: TIR domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermoflavifilum thermophilum (bacteria)
Molecular weightTheoretical: 53.256734 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
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 #2: Piwi domain-containing protein

MacromoleculeName: Piwi domain-containing protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermoflavifilum thermophilum (bacteria)
Molecular weightTheoretical: 58.304848 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
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 #3: guide RNA

MacromoleculeName: guide RNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Molecular weightTheoretical: 6.658989 KDa
SequenceString:
AAACGGCUCU AAUCUAUUAG U

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Macromolecule #4: target ssDNA

MacromoleculeName: target ssDNA / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Molecular weightTheoretical: 7.680997 KDa
SequenceString:
(DC)(DA)(DA)(DC)(DT)(DA)(DA)(DT)(DA)(DG) (DA)(DT)(DT)(DA)(DG)(DA)(DG)(DC)(DC)(DG) (DT)(DT)(DT)(DA)(DT)

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: RANDOM ASSIGNMENT
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.54 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 615987

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