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Updated: Sep 6, 2025
Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography
Published on: September 14, 2014
Method for the structural analysis of Twinkle mitochondrial DNA helicase by cryo-EM
Amanda A Riccio1, Jonathan Bouvette2, Matthew J Longley1
1Mitochondrial DNA Replication Group, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709, USA.
Abstract:
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.
Insights
Researchers developed a protocol to determine the structure of the Twinkle helicase, a key protein in mitochondrial DNA replication. This work provides the first high-resolution structure of this vital enzyme.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Mitochondrial DNA (mtDNA) replication relies on the mitochondrial replisome, a complex protein machinery.
- The Twinkle helicase, a Superfamily 4 (SF4) helicase, is crucial for maintaining mitochondrial genome integrity.
- While general SF4 helicase mechanisms are known, specific molecular details for mitochondrial Twinkle remain elusive.
Purpose of the Study:
- To establish a protocol for the expression, purification, and structural analysis of the Twinkle helicase.
- To obtain the first high-resolution structure of the W315L clinical variant of Twinkle helicase.
- To provide a foundation for future structural studies of Twinkle and other SF4 helicases.
Main Methods:
- Protein expression and purification of the Twinkle helicase W315L variant.
- Single-particle cryo-electron microscopy (cryo-EM) for structural determination.
- Development of an adaptable protocol for high-resolution structural studies.
Main Results:
- Successful expression and purification of the Twinkle helicase W315L variant.
- Determination of the first high-resolution cryo-EM structure of Twinkle helicase.
- Establishment of a reproducible protocol for structural studies.
Conclusions:
- The developed protocol enables high-resolution structural studies of the Twinkle helicase.
- The first high-resolution structure provides insights into the molecular mechanisms of mitochondrial DNA replication.
- This work facilitates future research on Twinkle helicase and related SF4 helicases.
