Rubinstein Lab
Rubinstein Lab
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Research

Summary

The 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 usually occurs at the level of developing new algorithms and software for image analysis and performing calculations with images and models of molecular structure.

Cryo-EM

Electron cryomicroscopy (cryo-EM) of macromolecular assemblies has become an important technique in structural biology. The method allows biologists to bridge the resolution gap between images of cells from light microscopy and conventional electron microscopy and the high-resolution information available from X-ray crystallography and NMR spectroscopy.

Figure of ATP synthase
Figure 1. A 3-D model of the ATP synthase from cryo-EM with an atomic model of the F1-c10 subcomplex docked into it. From Rubinstein et al., 2003.

ATP synthase

The ATP synthase is the central enzyme in biological energy metabolism. Our work aims to combine cryo-EM and structures derived from X–ray crystallography to build an empirical model of intact ATP synthase. Aspects of this project involve a close collaboration with the research group Sir John Walker (MRC Mitochondrial Biology Unit, Cambridge, UK). The ATP synthase is of medical interest because it presents a target for the generation of specific inhibitors that may serve as therapeutic agents for the treatment of bacterial infections, ischemia-reperfusion injury and some cancers.

V-ATPase

The vacuolar-type ATPase (V-ATPase) is responsible for pumping protons across a membrane to generate a proton motive force in many cellular contexts. We have begun cryo-EM studies of the bacterial complex, which is actually used for ATP synthesis, and the eukaryotic complex. The V-ATPase has an important role in several diseases, including osteoporosis (where V-ATPase is used to dissolve bone minerals) and cancer (where extracellular acidification by V-ATPase helps tumours to metastasize and invade surrounding tissues).

Collaborative projects

There are several collaborative projects underway in the research group. These projects, done in collaboration with established investigators, are pursued due to their biological importance and because the data that they yield will help to drive methodological developments.

Method development

Cryo-EM is an evolving method with great unexplored potential for high-resolution structure determination and the investigation of conformational changes and dynamic complexes. New techniques that will be developed, in part in this laboratory, will expand the potential of cryo-EM to address questions about macromolecular assemblies that currently frustrate crystallographic and spectroscopic approaches. The understanding of molecular processes in biology has often been tightly coupled to the development of new methods. In this tradition, our research program is a composite of the investigation of systems of fundamental biological importance with the development of electron cryomicroscopy methods. Through this combination, we intend to solve problems of importance to biology and medicine and expand our potential to address new questions in structural biology.

A number of new methods have been developed in the lab. If you are interested in any of the software that we have developed, please see the “software” page of this website. Tools include the rotational analysis approach for determining angular orientations of molecules.

Figure showing rotational analysis
Figure 2, A. The rotational analysis method allows the relative orientations of side-views of a molecule to be assigned in order to build an initial model of the complex.
Figure showing Frehand test
Figure 2, B. Optimized weighting of the phase residual function used to align particles leads to improved alignments, as shown by the Freehand test described by Rosenthal and Henderson (2003).
Funding
Research in the lab is funded by The Hospital for Sick Children, The Canadian Institutes of Health Research, the Canadian Foundation for Innovation, and the Ontario Ministry of Research and Innovation.