The vast majority of intracellular communication occurs through soluble protein complexes and membrane proteins. The study of these types of molecules has long been the domain of X-ray crystallography (for stable, crystallizable proteins) or NMR (for peptides and small proteins). However, recent advances in detectors and image processing have made the possibility of high resolution – or even atomic-resolution – structure determination by cryo-electron microscopy a reality.
One of the first membrane protein structures that we determined was of the E. coli bacterial chemotaxis receptor Tsr. More recently, we have focused on using tomographic and single particle methods to determine structures of membrane proteins like the AMPA glutamate receptor GluK2 and its relative GluA2 in resting, activated and desensitized states. These were the first high-resolution structures of the full-length, native GluK2 receptor, revealing a structural mechanism for glutamate receptor channel opening and desensitization.
We have also applied similar techniques to the bacterial magnesium-gated channel CorA, a small, pentameric complex. X-ray crystallographic studies showed that when the channel is bound with high levels of magnesium, it adopts a closed, 5-fold symmetric conformation. Using single particle analysis, we showed that, as the channel transitions to low magnesium conditions, the complex assumes a series of asymmetric conformations, with the cytoplasmic domains splaying out in a way that reoganizes the transmembrane domain and opens the pore. This type of analysis was only possible with cryo-electron microscopy, which can be used to resolve multiple conformations present in a single sample.
Another key area of interest has been to locate small molecules, such as nucleotides or drugs, within binding pockets of enzymatic complexes. One complex of particular interest is the AAA ATPase p97, a target for cancer therapeutic development due to its role in the proteasomal pathway. Because of its flexibility, the full p97 complex has not been amenable to study by X-ray crystallography; further, although individual domains of the protein have been crystallized, no single domain could be shown to be structurally affected by drugs that potently inhibited enzymatic activity. Using cryo-EM single particle analysis, we were able not only to demonstrate a variety of structural conformations of the p97 complex during the ATPase cycle, we further were able to directly show density for drug bound at the junction between two domains of the complex, demonstrating that the drug blocks the protein’s ratchet-type motion.