8EA3

V-K CAST Transpososome from Scytonema hofmanni, major configuration

Summary for 8EA3

• Entry DOI: 10.2210/pdb8ea3/pdb
• EMDB information: 27971 27972
• Descriptor: Target_LE, RE_F, RE_R1, ... (14 entities in total)
• Functional Keywords: dna binding protein, dna binding protein-dna-rna complex, dna binding protein/dna/rna
• Biological source: Scytonema hofmannii; More
• Total number of polymer chains: 30
• Total formula weight: 1230345.09

Authors

Rizo, A.N.,Park, J.-U.,Tsai, A.W.,Kellogg, E.H. (deposition date: 2022-08-27, release date: 2022-11-30, Last modification date: 2024-06-19)

Primary citation

Park, J.U.,Tsai, A.W.,Rizo, A.N.,Truong, V.H.,Wellner, T.X.,Schargel, R.D.,Kellogg, E.H.
Structures of the holo CRISPR RNA-guided transposon integration complex.
Nature, 613:775-782, 2023

PubMed Abstract: CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism. CAST elements contain multiple conserved proteins: a CRISPR effector (Cas12k or Cascade), a AAA+ regulator (TnsC), a transposase (TnsA-TnsB) and a target-site-associated factor (TniQ). These components are thought to cooperatively integrate DNA via formation of a multisubunit transposition integration complex (transpososome). Here we reconstituted the approximately 1 MDa type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined its structure using single-particle cryo-electon microscopy. The architecture of this transpososome reveals modular association between the components. Cas12k forms a complex with ribosomal subunit S15 and TniQ, stabilizing formation of a full R-loop. TnsC has dedicated interaction interfaces with TniQ and TnsB. Of note, we observe TnsC-TnsB interactions at the C-terminal face of TnsC, which contribute to the stimulation of ATPase activity. Although the TnsC oligomeric assembly deviates slightly from the helical configuration found in isolation, the TnsC-bound target DNA conformation differs markedly in the transpososome. As a consequence, TnsC makes new protein-DNA interactions throughout the transpososome that are important for transposition activity. Finally, we identify two distinct transpososome populations that differ in their DNA contacts near TniQ. This suggests that associations with the CRISPR effector can be flexible. This ShCAST transpososome structure enhances our understanding of CAST transposition systems and suggests ways to improve CAST transposition for precision genome-editing applications.

PubMed: 36442503
DOI: 10.1038/s41586-022-05573-5

Experimental method

ELECTRON MICROSCOPY (3.7 Å)