High-resolution structural biology – that is, imaging and analysis of proteins and macromolecular complexes at resolutions that allow one to draw conclusions about structural mechanism – has long been the province of techniques like X-ray crystallography and NMR. However, it is now possible to achieve these types of resolutions by cryo-electron microscopy, which does not require crystallization, and can be applied to complexes large and small alike. At the Center for Molecular Microscopy, our cryo-EM section is focused on determining structures of proteins and macromolecular complexes to high resolution.
Although electron microscopy has been used to image biological materials for decades, the use of cryogenic temperatures, which reduces the damage from electrons during imaging, allows for the study of unfixed, unstained proteins and molecular complexes. Nevertheless, even with the protection of cryogenic temperatures, the electron dose still needs to be very low in order to avoid damaging sensitive biological samples.
Within the last few years, several key advances have allowed for better and better resolution: first, a new type of camera (direct electron detectors) has been introduced, which allows for much better signal detection; and second, more computing power coupled with new algorithms for processing images has allowed researchers to tease more information out of existing electron microscopic images. For the first time, it is possible to acquire near-atomic resolution information from cryo-electron microscopy.
At the Center for Molecular Microscopy, we use both single particle analysis with cryo-electron microscopy, as well as cryo-electron tomography coupled with sub-tomogram averaging, to determine structures of protein complexes.
Areas of Biological Research Interest
Recent cryo-EM work in the laboratory has focused on three general areas: the structure of small soluble protein complexes, structure of membrane proteins, and the structure of proteins displayed on the surface of viruses.
For the last several years, we have been studying the envelope glycoprotein “entry spike” of HIV-1, and its relative SIV, by cryo-electron microscopy, resulting in several key structures of this protein complex. Early work on the HIV and SIV envelope glycoproteins (Env) was done using cryo-electron tomography of whole viruses, producing a host of structures of the Env spike in various conformations, and bound by a number of neutralizing antibodies. More recently, we used single particle analysis to determine higher resolution structures of soluble versions of the entry spike, reporting the presence of a trio of helices at the base of the complex.
We have also begun to use newly available detectors and reconstruction algorithms to determine the structures of soluble and integral membrane complexes to high resolution. Two recent examples of this include the ionotropic glutamate receptor, an ion channel with three distinct conformations, and the small protein complex beta-galactosidase, which we determined to a resolution of 3.5 angstroms.
Going forward, we are particularly interested in applying these technologies to a variety of protein complexes, including but not limited to those involved in cancer signaling, ion channels and other membrane proteins, as well as protein complexes involved in cellular metabolism.