Cryo-Electron microscopy of cardiac thin filaments

The main components of cardiac thin filaments are actin and the regulatory proteins troponin and tropomyosin which confer Calcium sensitivity. All of these proteins can carry mutations related to heart disease. (Marques and de Oliveira, 2016) To understand the process of muscle regulation and the effects of mutations at a molecular level we need to determine high resolution structures of this macromolecular complex.

Cryo-Electron microscopy (Cryo-EM) provides a means of achieving sub-nanometre 3D structures of these filaments that cannot be crystallised. The recent ‘resolution revolution’ (Callaway, 2015) means that atomic structures are now achievable through Cryo-EM. The acquisition of a state of the art microscope at Bristol and our experience in image processing the thin filament data puts us in an excellent position determine atomic models of the thin filament.

We have successfully isolated zebrafish cardiac thin filaments, a major model for the study of human cardiac disease (Gut et al., 2017) and located the regulatory protein troponin in this data which will allow us to employ our novel image processing procedures (Paul et al., 2010).

Aims
Our aim is to understand the process of muscle regulation and the effects of mutations at a molecular level. 3D atomic models of the thin filament in different functional states will provide insight into the mechanism of regulation (Paul et al., 2017) in addition, we intend to look at the difference in the secondary structure of the proteins caused by mutations that are known to cause cardio myopathies.