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- PDB-8u72: Cryo-EM structure of the SPARTA oligomer with guide RNA and target DNA -

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Basic information

Entry
Database: PDB / ID: 8u72
TitleCryo-EM structure of the SPARTA oligomer with guide RNA and target DNA
Components
  • DNA (5'-D(P*TP*AP*TP*AP*CP*AP*AP*CP*CP*TP*AP*CP*TP*AP*CP*CP*TP*C)-3')
  • Piwi domain-containing protein
  • RNA (5'-R(P*UP*GP*AP*GP*GP*UP*AP*GP*UP*AP*GP*GP*UP*UP*GP*UP*AP*UP*A)-3')
  • TIR domain-containing protein
KeywordsRNA BINDING PROTEIN/RNA/DNA / RNA / DNA / Argonaute / TIR / immune system / RNA BINDING PROTEIN-RNA-DNA complex
Function / homology
Function and homology information


nucleic acid binding / signal transduction
Similarity search - Function
TIR domain / Toll/interleukin-1 receptor homology (TIR) domain / Toll/interleukin-1 receptor homology (TIR) domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / Piwi domain-containing protein / TIR domain-containing protein
Similarity search - Component
Biological speciesThermoflavifilum thermophilum (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsMalik, R. / Kottur, J. / Aggarwal, A.K.
Funding support United States, 1items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 12, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
U: RNA (5'-R(P*UP*GP*AP*GP*GP*UP*AP*GP*UP*AP*GP*GP*UP*UP*GP*UP*AP*UP*A)-3')
V: DNA (5'-D(P*TP*AP*TP*AP*CP*AP*AP*CP*CP*TP*AP*CP*TP*AP*CP*CP*TP*C)-3')
H: TIR domain-containing protein
I: Piwi domain-containing protein
E: Piwi domain-containing protein
M: RNA (5'-R(P*UP*GP*AP*GP*GP*UP*AP*GP*UP*AP*GP*GP*UP*UP*GP*UP*AP*UP*A)-3')
N: DNA (5'-D(P*TP*AP*TP*AP*CP*AP*AP*CP*CP*TP*AP*CP*TP*AP*CP*CP*TP*C)-3')
S: RNA (5'-R(P*UP*GP*AP*GP*GP*UP*AP*GP*UP*AP*GP*GP*UP*UP*GP*UP*AP*UP*A)-3')
T: DNA (5'-D(P*TP*AP*TP*AP*CP*AP*AP*CP*CP*TP*AP*CP*TP*AP*CP*CP*TP*C)-3')
X: RNA (5'-R(P*UP*GP*AP*GP*GP*UP*AP*GP*UP*AP*GP*GP*UP*UP*GP*UP*AP*UP*A)-3')
Y: DNA (5'-D(P*TP*AP*TP*AP*CP*AP*AP*CP*CP*TP*AP*CP*TP*AP*CP*CP*TP*C)-3')
D: TIR domain-containing protein
B: TIR domain-containing protein
C: Piwi domain-containing protein
F: TIR domain-containing protein
G: Piwi domain-containing protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)528,40720
Polymers528,31016
Non-polymers974
Water1,02757
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 8 molecules HDBFIECG

#3: Protein
TIR domain-containing protein / TIR-APAZ


Mass: 53256.734 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermoflavifilum thermophilum (bacteria)
Gene: SAMN05660895_1670 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1I7NFG5
#4: Protein
Piwi domain-containing protein / pAgo


Mass: 58304.848 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermoflavifilum thermophilum (bacteria)
Gene: SAMN05660895_1671 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1I7NFD7

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RNA chain / DNA chain , 2 types, 8 molecules UMSXVNTY

#1: RNA chain
RNA (5'-R(P*UP*GP*AP*GP*GP*UP*AP*GP*UP*AP*GP*GP*UP*UP*GP*UP*AP*UP*A)-3')


Mass: 6812.045 Da / Num. of mol.: 4 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: DNA chain
DNA (5'-D(P*TP*AP*TP*AP*CP*AP*AP*CP*CP*TP*AP*CP*TP*AP*CP*CP*TP*C)-3')


Mass: 13703.841 Da / Num. of mol.: 4 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 61 molecules

#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 57 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: SPARTA complex with gRNA/tDNA / Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Source (natural)Organism: Thermoflavifilum thermophilum (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: YES / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
EM embeddingMaterial: Vitreous ice
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 51.11 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 238432 / Details: Composite cryo-EM map / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00330693
ELECTRON MICROSCOPYf_angle_d0.58242350
ELECTRON MICROSCOPYf_dihedral_angle_d12.8995284
ELECTRON MICROSCOPYf_chiral_restr0.0424854
ELECTRON MICROSCOPYf_plane_restr0.0054797

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