In the last few years, there has been a critical shift in the cryo-electron microscopy field, bringing near-atomic resolution structural analysis of small protein complexes into the realm of possibility for the first time. This year we have made significant progress in applying new methods and technologies to our work both on membrane proteins and on small protein complexes. Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the vertebrate brain. To better understand how structural changes gate ion flux across the membrane, we trapped AMPA and kainate receptor subtypes in their major functional states and analyzed the resulting structures using cryo-electron microscopy. We show that transition to the active state involves a corkscrew motion of the receptor assembly, driven by closure of the ligand binding domain. Desensitization is accompanied by rupture of the amino terminal domain tetramer in AMPA, but not kainate receptors, with a 2-fold to 4-fold symmetry transition in the ligand binding domains in both subtypes. A 7.6 Angstrom structure of a desensitized kainate receptor GluK2 showed how these changes accommodate channel closing. Our study exploited the availability of unique AMPA receptor allosteric modulators to trap GluA2 in the active state, while for GluK2 the greater thermodynamic stability of the desensitized state combined with a more tightly bound ligand yielded a higher resolution structure than could be achieved for GluA2. These findings integrate previous physiological, biochemical, and structural analyses of glutamate receptors and provide a molecular explanation for key steps in receptor gating, and have provided a detailed glimpse into the overall gating cycle of glutamate receptors. We have continued our work on the AMPA and kainite receptors, specifically investigating new ways to solubilize and visualize integral membrane proteins using single particle analysis. We have also initiated systematic efforts to compare structures of the receptors when they are solubilized using different detergents as well as amphipol, a reagent commonly used to solubilize and stabilize membrane proteins for structural analysis. In addition to the iGluRs, we have also taken on structural studies of the ion channel CorA, in collaboration with Eduardo Perozo from the University of Illinois. The 200 kDa pentameric membrane channel CorA is the major Mg2+ uptake system in bacteria. CorA contributes to Mg2+ homeostasis through a negative feedback loop, where Mg2+ binding at the subunit interface leads to channel closure and low Mg2+ concentrations stabilize the open conformation. Several crystal structures have shed light on the architecture of the magnesium-bound closed state, while electron paramagnetic resonance (EPR) spectroscopic studies of purified CorA revealed large quaternary conformational changes associated with magnesium binding/unbinding. In continuing efforts to understand the mechanism of ion transport, we now have a map at 3.8 Angstrom resolution in the magnesium-bound state, and have determined a novel, asymmetric structure for the unbound state.
| Earl, Lesley A; Falconieri, Veronica; Subramaniam, Sriram (2018) Microbiology catches the cryo-EM bug. Curr Opin Microbiol 43:199-207 |
| Kang, Yanyong; Kuybeda, Oleg; de Waal, Parker W et al. (2018) Cryo-EM structure of human rhodopsin bound to an inhibitory G protein. Nature 558:553-558 |
| Kang, Yanyong; Kuybeda, Oleg; de Waal, Parker W et al. (2018) Publisher Correction: Cryo-EM structure of human rhodopsin bound to an inhibitory G protein. Nature : |
| Wohlbold, Teddy John; Podolsky, Kira A; Chromikova, Veronika et al. (2017) Broadly protective murine monoclonal antibodies against influenza B virus target highly conserved neuraminidase epitopes. Nat Microbiol 2:1415-1424 |
| Earl, Lesley A; Falconieri, Veronica; Milne, Jacqueline Ls et al. (2017) Cryo-EM: beyond the microscope. Curr Opin Struct Biol 46:71-78 |
| Meyerson, Joel R; Chittori, Sagar; Merk, Alan et al. (2016) Structural basis of kainate subtype glutamate receptor desensitization. Nature 537:567-571 |
| Subramaniam, Sriram; Earl, Lesley A; Falconieri, Veronica et al. (2016) Resolution advances in cryo-EM enable application to drug discovery. Curr Opin Struct Biol 41:194-202 |
| Stuart, David I; Subramaniam, Sriram; Abrescia, Nicola G A (2016) The democratization of cryo-EM. Nat Methods 13:607-8 |
| Matthies, Doreen; Dalmas, Olivier; Borgnia, Mario J et al. (2016) Cryo-EM Structures of the Magnesium Channel CorA Reveal Symmetry Break upon Gating. Cell 164:747-56 |
| Subramaniam, Sriram; Kühlbrandt, Werner; Henderson, Richard (2016) CryoEM at IUCrJ: a new era. IUCrJ 3:3-7 |
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