Grant: CIHR Fellowship

Glutamate receptors are attractive targets for drug therapy as their deregulation is associated with Fragile X mental retardation, epilepsy, schizophrenia, depression, and neuropathic pain. One of the most abundant receptor of the nervous system is the AMPA glutamate receptor. It is a cation-selective ion channel which opens upon binding of the excitatory neurotransmitter glutamate. The study of this receptor complex in different functional forms (closed, open, and desensitized) is complicated by the presence of conformational heterogeneity, notably in the desensitized state. Nevertheless, obtaining high-resolution structures of this state could clarify the desensitization mechanism and regulation of glutamate receptors. Few experimental techniques are available to solve the atomic structure of large protein complexes in the presence of conformational heterogeneity. In the proposed project, we want to extend the methodology of cryo-electron tomography (cryoET) to study heterogeneous protein complexes at near-atomic resolution. CryoET provides geometrical information and can image one-of-a-kind 3D objects such as intact biological assemblies in near-physiological conditions. Improvements to this technique could lead to a fast, high-resolution structure determination method for a broader range of specimens. Our first objective is to improve the resolution of cryoET images for membrane complexes. We will use direct electron detectors to correct for sample movement, and new sampling schemes to reduce radiation damage. The second objective is to develop data processing algorithms for the treatment of sample heterogeneity. We will use two strategies: the simultaneous determination of the particle class and particle orientation, and the treatment of rigid protein domains as independent sub-particles. The third objective is to investigate the relationship between the structural heterogeneity of the GluA2 receptor complex and its dynamics observed by solid-state NMR.