Summary information and primary citation

PDB-id
8it1; SNAP-derived features in text and JSON formats; DNAproDB
Class
DNA binding protein-DNA-RNA
Method
cryo-EM (3.41 Å)
Summary
cryo-EM structure of crt-sparta-grna-tDNA tetramer (nadase active form)
Reference
Gao X, Shang K, Zhu K, Wang L, Mu Z, Fu X, Yu X, Qin B, Zhu H, Ding W, Cui S (2024): "Nucleic-acid-triggered NADase activation of a short prokaryotic Argonaute." Nature, 625, 822-831. doi: 10.1038/s41586-023-06665-6.
Abstract
Argonaute (Ago) mediates RNA or DNA guided inhibition of nucleic acids1,2. Although the mechanisms underlying eukaryotic (eAgos) and long prokaryotic Ago (pAgos) proteins are known, that of short pAgos remains elusive. Here, we determined cryo-EM structures of short pAgo and the associated TIR-APAZ proteins (SPARTA) from Crenotalea thermophila (Crt): a free-state Crt-SPARTA (3.27 Å), a guide RNA / target DNA loaded Crt-SPARTA (3.27 Å), two Crt-SPARTA dimers with distinct TIR organization (3.49 Å and 3.50 Å), and a Crt-SPARTA tetramer (3.41 Å). These structures reveal that the Crt-SPARTA is composed of a bilobal-fold Ago lobe connecting with a TIR lobe. Whereas the Crt-Ago harbors a MID and a PIWI domains, Crt-TIR-APAZ harbors a TIR, a N-like, a Linker and a Trigger domains. 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 acts as a roadblock preventing the guide RNA 5'- and the target DNA 3'-ends from reaching their canonical pockets, which 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 assemble into two parallel, head-to-tail TIR organization, indicating a NADase-active conformation, which is supported by our mutagenesis study. Our results reveal the structural basis of short pAgos in defensing invading nucleic acids and provide insights for optimizing SPARTA-based programmable DNA sequences detection.

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