Electron cryomicroscopy of macromolecular assemblies

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Research Interests

• Structural biology and electron cryomicroscopy
• Structure and function of macromolecular assemblies
• Membrane protein complexes
• Bioenergetics

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.

ATP synthase
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 Dunn Human Nutrition 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 angiogenesis in cancer.

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.

TonB dependent nutrient transport
Gram-negative bacteria couple the import of nutrients such as iron and vitamin B12 across their outer membrane to the proton motive force across their cytoplasmic membrane. This process depends on the soluble TonB protein and membrane bound ExbB-ExbD protein complex. Through a collaboration with Prof. Mirek Cygler (NRC-BRI, Montréal) we are using EM to study the structure of the membrane bound ExbB-ExbD complex. Understanding nutrient import in Gram-negative bacteria, which include /E. coli/ and the /P. aeruginosa/ (a major opportunistic pathogen for people with burns or cystic fibrosis), is essential for developing therapies against infection by these organisms.

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.

External Funding
Research in the laboratory is funded by The Hospital for Sick Children and operating grants from the Canadian Institutes of Health Research. A state-of-the-art cryo-EM facility was recently established at The Hospital for Sick Children with a Leaders Opportunity Fund award to Dr. Rubinstein from the Canadian Foundation for Innovation.

Bueler SA, Rubinstein JL. (2008). Location of subunit d in the peripheral stalk of the ATP synthase from Saccharomyces cerevisiae. Biochemistry 47, 11804-11810.

Lau WCY, Baker LA, Rubinstein JL. (2008). Cryo-EM structure of the yeast ATP synthase.Journal of Molecular Biology 382, 1256-64.

Sprangers R, Liqq X, Rubinstein JL, Schimmer AD, Kay LE. (2008). TROSY-based NMR evidence for a novel class of 20S proteasome inhibitor. Biochemistry, 47, 6727-34.

Baker LA, Rubinstein JL. (2008) Angle determination for side views in single particle electron microscopy. Journal of Structural Biology 162, 260-270.

Andersen F, Knudsen B, Oliveira C, Frahlich R, Kruger D, Bungert J, McKenna M, McKenna R, Juul S, Veigaard C, Koch J, Rubinstein JL, Guldbrandtsen B, Hede M, Karlsson G, Andersen A, Pedersen J, Knudsen B. (2008). Assembly and structural analysis of a covalently closed nano-scale DNA cage. Nucleic Acids Research 36, 1113-9.

Tocilj A, Munger C, Morona R, Purins L, Ajamian E, Wagner J, Papadopoulos M, Van Den Bosch L, Rubinstein JL, Proteau A, Féthière J, Matte A, Cygler M. (2008). Bacterial polysaccharide co-polymerases share a common framework for control of polymer length.Nature Structural and Molecular Biology 15, 130-138.

Tate CG, Rubinstein JL. (2008). Structure determination of membrane proteins by electron cryo-microscopy. In Biophysical Analysis of Membrane Proteins: Investigating Structure and Function. Eva Pebay-Peyroula (Editor). Wiley InterScience. Hoboken, NJ, USA.

Rubinstein JL. (2007). Structural analysis of membrane protein complexes by single particle electron microscopy. Methods 41, 409 - 416.

Edmonds L, Liu A, Kwan JJ, Avanessy A, Caracoglia M, Yang I, Maxwell KL, Rubinstein JL, Davidson AR, Donaldson LW. (2007) The NMR structure of the gpU tail-terminator protein from bacteriophage lambda: identification of sites contributing to Mg(II)-mediated oligomerization and biological function. Journal of Molecular Biology 365, 175 - 186.

Rubinstein, J. L., Dickson, V. K., Runswick, M. J. and Walker, J. E. (2005). ATP synthase from Saccharomyces cerevisiae: Location of the subunit h in the peripheral stalk region. Journal of Molecular Biology 345:513-520.

Rubinstein, J. L., Walker, J. E. and Henderson, R. (2003). Structure of the mitochondrial ATP synthase by electron cryomicroscopy. EMBO Journal 22:6182 – 6192.[See “Faculty of 1000” (www.facultyof1000.com)].

Rubinstein, J. L., Holt, L. J., Walker, J. E. and Tomlinson, I. M. (2003). Use of phage display and high-density screening for the isolation of an antibody against the 51 kDa subunit of complex I. Analytical Biochemistry 314:294 – 300.

Rubinstein, J. L. and Walker, J. E. (2002). ATP synthase from Saccharomyces cerevisiae: Location of the OSCP subunit in the peripheral stalk region. Journal of Molecular Biology 231:613 - 619.