8YWH
Cryo-EM structure of small and dead form SaCas9-RNA-DNA ternary complex (sdCas9)
Summary for 8YWH
| Entry DOI | 10.2210/pdb8ywh/pdb |
| Related | 5AXW 7VW3 |
| EMDB information | 39633 |
| Descriptor | sCas9 (Compact SaCas9), sgRNA, Target DNA, ... (4 entities in total) |
| Functional Keywords | crispr/cas9, thermostable protein engineering, domain minimized cas, engineered sacas9, dna binding protein |
| Biological source | Staphylococcus aureus More |
| Total number of polymer chains | 4 |
| Total formula weight | 172047.02 |
| Authors | Kang, E.S.,Kim, N.H.,Thach, T.T.,Hyun, J.,Kim, Y.H. (deposition date: 2024-03-31, release date: 2025-04-02) |
| Primary citation | Kang, E.S.,Kim, N.H.,Lim, H.K.,Jeon, H.,Han, K.,No, Y.H.,Kim, K.,Khaleel, Z.H.,Shin, D.,Eom, K.,Nam, J.,Lee, B.S.,Kim, H.J.,Suh, M.,Lee, J.,Thach, T.T.,Hyun, J.,Kim, Y.H. Structure-Guided Engineering of Thermodynamically Enhanced SaCas9 for Improved Gene Suppression. Adv Mater, :e2404680-e2404680, 2024 Cited by PubMed Abstract: Proteins with multiple domains play pivotal roles in various biological processes, necessitating a thorough understanding of their structural stability and functional interplay. Here, a structure-guided protein engineering approach is proposed to develop thermostable Cas9 (CRISPR-associated protein 9) variant for CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference applications. By employing thermodynamic analysis, combining distance mapping and molecular dynamics simulations, deletable domains are identified to enhance stability while preserving the DNA recognition function of Cas9. The resulting engineered Cas9, termed small and dead form Cas9, exhibits improved thermostability and maintains target DNA recognition function. Cryo-electron microscopy analysis reveals structural integrity with reduced atomic density in the deleted domain. Fusion with functional elements enables intracellular delivery and nuclear localization, demonstrating efficient gene suppression in diverse cell types. Direct delivery in the mouse brain shows enhanced knockdown efficiency, highlighting the potential of structure-guided engineering to develop functional CRISPR systems tailored for specific applications. This study underscores the significance of integrating computational and experimental approaches for protein engineering, offering insights into designing tailored molecular tools for precise biological interventions. PubMed: 38944889DOI: 10.1002/adma.202404680 PDB entries with the same primary citation |
| Experimental method | ELECTRON MICROSCOPY (2.97 Å) |
Structure validation
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