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- PDB-8isy: Cryo-EM structure of free-state Crt-SPARTA -

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Basic information

Entry
Database: PDB / ID: 8isy
TitleCryo-EM structure of free-state Crt-SPARTA
Components
  • Piwi domain-containing protein
  • TIR domain-containing protein
KeywordsDNA BINDING PROTEIN / Ago / DNA/RNA
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.27 Å
AuthorsGao, X. / Shang, K. / Zhu, K. / Wang, L. / Mu, Z. / Fu, X. / Yu, X. / Qin, B. / Zhu, H. / Ding, W. / Cui, S.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nature / Year: 2024
Title: Nucleic-acid-triggered NADase activation of a short prokaryotic Argonaute.
Authors: Xiaopan Gao / Kun Shang / Kaixiang Zhu / Linyue Wang / Zhixia Mu / Xingke Fu / Xia Yu / Bo Qin / Hongtao Zhu / Wei Ding / Sheng Cui /
Abstract: Argonaute (Ago) proteins mediate RNA- or DNA-guided inhibition of nucleic acids. Although the mechanisms used by eukaryotic Ago proteins and long prokaryotic Ago proteins (pAgos) are known, that used ...Argonaute (Ago) proteins mediate RNA- or DNA-guided inhibition of nucleic acids. Although the mechanisms used by eukaryotic Ago proteins and long prokaryotic Ago proteins (pAgos) are known, that used by short pAgos remains elusive. Here we determined the cryo-electron microscopy structures of a short pAgo and the associated TIR-APAZ proteins (SPARTA) from Crenotalea thermophila (Crt): a free-state Crt-SPARTA; a guide RNA-target DNA-loaded Crt-SPARTA; two Crt-SPARTA dimers with distinct TIR organization; and a Crt-SPARTA tetramer. These structures reveal that Crt-SPARTA is composed of a bilobal-fold Ago lobe that connects with a TIR lobe. Whereas the Crt-Ago contains a MID and a PIWI domain, Crt-TIR-APAZ has a TIR domain, an N-like domain, a linker domain and a trigger domain. The bound RNA-DNA duplex adopts a B-form conformation that is recognized by base-specific contacts. Nucleic acid binding causes conformational changes because the trigger domain acts as a 'roadblock' that prevents the guide RNA 5' ends and the target DNA 3' ends from reaching their canonical pockets; this disorders the MID domain and promotes Crt-SPARTA dimerization. Two RNA-DNA-loaded Crt-SPARTA dimers form a tetramer through their TIR domains. Four Crt-TIR domains assemble into two parallel head-to-tail-organized TIR dimers, indicating an NADase-active conformation, which is supported by our mutagenesis study. Our results reveal the structural basis of short-pAgo-mediated defence against invading nucleic acids, and provide insights for optimizing the detection of SPARTA-based programmable DNA sequences.
History
DepositionMar 21, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 18, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 8, 2023Group: Data collection / Structure summary / Category: audit_author / em_author_list / struct / Item: _struct.title
Revision 1.2Feb 7, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Piwi domain-containing protein
B: TIR domain-containing protein


Theoretical massNumber of molelcules
Total (without water)111,5622
Polymers111,5622
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Piwi domain-containing protein / CrtAgo


Mass: 58304.848 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermoflavifilum thermophilum (bacteria)
Gene: SAMN05660895_1671 / Production host: Expression vector pET-mod (others) / References: UniProt: A0A1I7NFD7
#2: Protein TIR domain-containing protein / CrtTIR-APAZ


Mass: 53256.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermoflavifilum thermophilum (bacteria)
Gene: SAMN05660895_1670 / Production host: Expression vector pET-mod (others) / References: UniProt: A0A1I7NFG5

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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: prokaryotic Argonaute System / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Thermoflavifilum thermophilum (bacteria)
Source (recombinant)Organism: Expression vector pET-mod (others)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil R1.2/1.3
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 FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 1800 nm / Calibrated defocus min: 1800 nm / Calibrated defocus max: 2500 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 66 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
7PHENIX1.20.1model fitting
9PHENIX1.20.1model refinement
13cryoSPARC4.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.27 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1158576 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0048071
ELECTRON MICROSCOPYf_angle_d0.65810905
ELECTRON MICROSCOPYf_dihedral_angle_d3.2391036
ELECTRON MICROSCOPYf_chiral_restr0.0481171
ELECTRON MICROSCOPYf_plane_restr0.0041387

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