9ECA
Intermediate state of wild-type EsCas13d ternary complex with C4A mismatch
Summary for 9ECA
| Entry DOI | 10.2210/pdb9eca/pdb |
| EMDB information | 47904 |
| Descriptor | EsCas13d, crRNA, Target RNA, ... (4 entities in total) |
| Functional Keywords | cas13, crispr, hepn, rna nuclease, rna binding protein-rna complex, rna binding protein/rna |
| Biological source | [Eubacterium] siraeum DSM 15702 More |
| Total number of polymer chains | 3 |
| Total formula weight | 137229.36 |
| Authors | Chou, C.W.,Finkelstein, I.J. (deposition date: 2024-11-13, release date: 2025-11-19, Last modification date: 2026-09-02) |
| Primary citation | Chou, C.W.,Sinan, S.,Kuo, H.C.,Chang, Y.C.,Arguello, C.,Sahaya, D.,Russell, R.,Finkelstein, I.J. Structural basis for target discrimination and activation by Cas13d. Sci Adv, 12:eaec4221-eaec4221, 2026 Cited by PubMed Abstract: CRISPR-Cas13d is increasingly used for RNA knockdowns, but off-target cleavage of near-cognate RNAs hinders its broader adoption. Here, we solve seven cryo-electron microscopy structures of wild-type Cas13d in complex with matched and mismatched targets. These structures reveal active, intermediate, and inactive states that illustrate a detailed activation mechanism. Upon target RNA binding, the CRISPR RNA undergoes marked conformational changes. The Helical-1 domain transitions from a docked state with the amino-terminal domain to an allosterically switched conformation that stabilizes the RNA duplex. Quantitative kinetics show that a single proximal mismatch preserves the binding rate constant but abolishes nuclease activity by trapping Cas13d in an inactive state. We also identify an active site loop in the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains that regulates substrate accessibility and can be mutated to generate both hypo- and hyperactivated variants. These findings establish the structural basis for Cas13d mismatch surveillance and provide a framework for engineering HEPN nuclease specificity and activity. PubMed: 42616864DOI: 10.1126/sciadv.aec4221 PDB entries with the same primary citation |
| Experimental method | ELECTRON MICROSCOPY (3.19 Å) |
Structure validation
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