Unraveling biological mechanisms with cryo-electron microscopy

Introduction

Cryo-Electron Microscopy (EM) is a powerful technique to visualize biological processes. While many studies are centered on extending structural information to higher resolution, not much has been done in the field to examine how multiple players collectively form pathways and systems. In fact, many experiments are performed on macromolecular complexes biochemically removed from their proper cellular context. Our present molecular imaging platform permits us to capture and to view functional pathways within a single image.The use of our unique imaging technology opens up a new avenue for viewing biological mechanisms within a functionally relevant, near-native environment.

We utilize the bacterial protein synthesis machinery as a model system to develop new methodologies. The combined use of our Affinity Capture technology with single particle EM provide the basis for visualizing RNA-dependent pathways in a remarkable new way. The cryo-EM image to the left indicates a variety of different protein assemblies (dark particles) that are connected by native strands of RNA, scale bar is 15 nm. This demonstrates how large biological assemblies can be captured in real-time.

Just as our current molecular imaging system can be used to specifically isolate proteins of interest from within cells, we can also apply these methods at the level of whole cells. Cryo-Electron Tomography of whole cells is a newly evolving technique in the EM field that has never been used to view stem cells. This is primarily due to the thickness of whole cells and the inefficiency of applying them to an EM grid. Stem cells found in early development, however, are of a reasonable size for TEM imaging. The main challenge to overcome, therefore, lies in applying them to an EM grid. Our Affinity Capture approach works nicely to accomplish this task. Once cells, including cancer cells, are properly attached to our grids, we can examine them in 3D using cryo-Tomography.