Research

Our group studies the structure and function of macromolecular assemblies using electron cryomicroscopy (cryo-EM), image analysis, molecular biology, and molecular genetics. We also develop the tools of cryo-EM so that we can answer questions that are not amenable to the techniques that currently exist. This process occurs at the level of developing new algorithms and software for image analysis, performing calculations with images and models of molecular structure, and developing new methods for specimen preparation.

RESEARCH DIRECTIONS

Cryo-EM method development and Bioenergetics

Electron cryomicroscopy (cryo-EM) of macromolecular assemblies has become an important technique in structural biology. The method allows biologists to determine high-resolution structures of proteins that cannot be crystallized for X-ray crystallography, and also allows for investigation of the molecular motions that are essential for biomolecular function. We are developing new methods for preparing specimens, imaging them, and performing image analysis in order to expand the potential of the technique.

We are particularly interested in the control and flow of energy in biological systems (bioenergetics). In biology, chemical energy is frequently interconverted with transmembrane electrochemical gradients of protons. In humans and some pathogenic bacteria, the supply of energy from metabolism is dependent on the availability of the electron acceptor oxygen. Our structural studies aim to understand, at a molecular level, processes such as

  • mitochondrial oxidative phosphorylation (important in heart failure, neurobiology, and other areas)

  • mycobacteria oxidative phosphorylation (important as a target space for treating mycobacterial infections such as tuberculosis)

  • control of pH (important in the brain, skeleton, and immune system)

ATP synthase

The ATP synthase is the central enzyme in biological energy metabolism. Our work aims to determine high-resolution structures of the different types of ATP synthase, as well as understand how their dynamics facilitate their function. The mycobacterial ATP synthase is a validated drug target for the treatment of extensively drug-resistant tuberculosis. The eukaryotic ATP synthase may be responsible for a phenomenon known as the mitochondrial permeability transition that leads to cell death.

V-ATPase

Vacuolar-type ATPases are ATP-powered proton pumps that control the pH of numerous intracellular compartments in all eukaryotic cells and the extracellular environment of some specialized cells. These enzymes have important roles in endocytosis, exocytosis (including signal transmission in the brain), bone maintenance, kidney function, neurological disorders, and cancer.

Electron transport chain

The electron transport chain complexes establish the transmembrane proton motive force that powers ATP synthesis in mitochondria and aerobic bacteria such as the pathogenic mycobacteria that cause tuberculosis. Despite decades of study, it is unclear how the passage of electrons through the electron transport chain complexes, which ultimately leads to the reduction of oxygen to water, powers proton transport. Understanding this process will allow for improved targeting of the electron transport chain to treat diseases including mycobacterial infections.

Other

In addition to the projects listed above, there are a number of exciting new bioenergetics and collaborative directions being explored in the laboratory.

RESEARCH INFRASTRUCTURE

The laboratory is housed in the state-of-the art Peter Gilgan Centre for Research and Learning at the Hospital for Sick Children and is equipped with extensive infrastructure for cryo-EM. We are also involved in developing new hardware and testing new instruments developed by established manufacturers and startup companies.

  • A Thermo Fisher G3 Titan Krios with Falcon4 direct detector device camera

  • FEI Tecnai F20 with Gata K2 Summit direct detector device camera

  • 3 x Gatan 626 and Fischione 2550 cryospecimen holders

  • FEI Vitrobot grid freezing device

  • Leica EM GP2 grid freezing device

  • Gatan CP3 grid freezing device

  • 2 x PELCO EasiGlow glow discharge devices

  • Raise3D Pro 3D printer

We have a well-equipped wet/dry biochemistry laboratory with access to all standard biochemistry and biophysics equipment, and some not-so-standard equipment. We have experience and facilities for culturing of cells including Escherichia coli, Mycobacterium smegmatis, Helicobacter pylori, Legionella pneumophila, Thermus thermophilus, Saccharomyces cerevisiae, Pichia pastoris, and a variety of human cell lines.

Infrastructure for nano fabrication, including electron-beam lithography, is done at the Toronto Nanofabrication Centre