Thursday, March 22, 2012
“3D Cryo-Reconstruction of Icosahedral Viruses in Real Time”
Timothy S. Baker, Ph.D., Professor — Department of Chemistry & Biochemistry and Division of Biological Sciences, University of California, San Diego
The development of modern transmission electron cryo-microscopy and computer-based three-dimensional (3D) image reconstruction techniques has revolutionized how the structures of complex, biomacromolecular machines are studied. These and other correlative methods provide a pathway to understand how these machines function at the molecular level. However, this process is quite demanding and can be quite time consuming, and requires the successful completion of a large number of non-trivial steps. These include: specimen isolation, purification, and vitrification; low-dose TEM imaging; 3D structure determination; and visualization and interpretation of the final structure(s). Much attention has been devoted in recent years to simplifying and automating the imaging (e.g. with LEGINON) and structure determination (e.g. with APPION and AUTO3DEM) steps, and this automation has led to dramatic improvements in the resolutions that can be achieved in cryo-reconstruction work. It is now almost “routine” to achieve sub-nanometer resolution results with a wide variety of single-particle specimens. Concomitant with the dramatic improvements that have been achieved with automation to date, the time frame needed to go from “specimen” to “structure” has been drastically contracted from several man-years/months just a decade or so back to weeks/days or hours in highly favorable instances. Such technological breakthroughs now make it feasible to perform 3D reconstructions of symmetric, single particles (e.g. icosahedral viruses) at low-resolution (~20-25 Å) in a matter of minutes and sometimes seconds. Ultimately, the cryo-microscopist would like “instant” 3D feedback while the microscopy is performed, as this would, at a minimum, provide important data about the quality of the specimen as well as instrument performance, and such information would help dictate whether extensive imaging is warranted.
Results obtained with several different samples of icosahedral viruses will be highlighted to illustrate how close we have come to the goal of performing real time 3D at the microscope. Of course, numerous challenges remain and these will also be discussed.