Method for the structural analysis of Twinkle mitochondrial DNA helicase by cryo-EM.

Journal Article (Journal Article)

The mitochondrial replisome replicates the 16.6 kb mitochondria DNA (mtDNA). The proper functioning of this multicomponent protein complex is vital for the integrity of the mitochondrial genome. One of the critical protein components of the mitochondrial replisome is the Twinkle helicase, a member of the Superfamily 4 (SF4) helicases. Decades of research has uncovered common themes among SF4 helicases including self-assembly, ATP-dependent translocation, and formation of protein-protein complexes. Some of the molecular details of these processes are still unknown for the mitochondria SF4 helicase, Twinkle. Here, we describe a protocol for expression, purification, and single-particle cryo-electron microscopy of the Twinkle helicase clinical variant, W315L, which resulted in the first high-resolution structure of Twinkle helicase. The methods described here serve as an adaptable protocol to support future high-resolution studies of Twinkle helicase or other SF4 helicases.

Full Text

Duke Authors

Cited Authors

  • Riccio, AA; Bouvette, J; Longley, MJ; Krahn, JM; Borgnia, MJ; Copeland, WC

Published Date

  • September 2022

Published In

Volume / Issue

  • 205 /

Start / End Page

  • 263 - 270

PubMed ID

  • 35779765

Pubmed Central ID

  • PMC9398961

Electronic International Standard Serial Number (EISSN)

  • 1095-9130

Digital Object Identifier (DOI)

  • 10.1016/j.ymeth.2022.06.012


  • eng

Conference Location

  • United States