We have made significant progress towards using 3D cryo-electron microscopy to determine the structures of membrane proteins and ligand-induced conformational changes at resolutions of 20 Angstroms or better. Work on numerous membrane protein assemblies including chemotaxis receptors, multi-drug transporters and ion channels. The example below with glutamate receptors illustrates the strategy we are taking. Glutamate receptor ion channels are membrane proteins that mediate excitatory synaptic transmission in the central nervous system of vertebrates. Insight into molecular mechanisms underlying glutamate receptor gating is limited by lack of structural information for receptors trapped in different conformational states. Using single particle cryo-electron tomography, we determined the structures, at 20 Angstrom resolution, of full-length GluK2 kainate receptors trapped in antagonist-bound resting and agonist-bound desensitized states. The resting state, stabilized by the competitive antagonist LY466195, closely resembles the crystal structure of the AMPA receptor GluA2, with well-resolved proximal and distal subunits exhibiting crossover between the 2-fold symmetric amino terminal domain (ATD) and a 2-fold symmetric ligand binding domain (LBD) dimer of dimers assembly. In the desensitized state, the LBD undergoes a major rearrangement resulting in a separation of the four subunits by 25 Angstroms. However, the ATD, transmembrane and cytoplasmic regions of the receptor have similar conformations in the resting and desensitized states. The LBD rearrangement was not anticipated in prior models based on crystal structures for soluble LBD dimer assemblies, and we speculate that subunit separation allows a better match to the 4-fold symmetric ion channel domain. From fits of the ATD and LBD domains into the density map of the desensitized state, we have derived a structural model for differences in quaternary conformation between the resting and desensitized states.

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National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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Milne, Jacqueline L S; Borgnia, Mario J; Bartesaghi, Alberto et al. (2013) Cryo-electron microscopy--a primer for the non-microscopist. FEBS J 280:28-45
Schauder, David M; Kuybeda, Oleg; Zhang, Jinjin et al. (2013) Glutamate receptor desensitization is mediated by changes in quaternary structure of the ligand binding domain. Proc Natl Acad Sci U S A 110:5921-6
Kuybeda, Oleg; Frank, Gabriel A; Bartesaghi, Alberto et al. (2013) A collaborative framework for 3D alignment and classification of heterogeneous subvolumes in cryo-electron tomography. J Struct Biol 181:116-27
Hirai, Teruhisa; Subramaniam, Sriram; Lanyi, Janos K (2009) Structural snapshots of conformational changes in a seven-helix membrane protein: lessons from bacteriorhodopsin. Curr Opin Struct Biol 19:433-9
Bartesaghi, Alberto; Subramaniam, Sriram (2009) Membrane protein structure determination using cryo-electron tomography and 3D image averaging. Curr Opin Struct Biol 19:402-7
Milne, Jacqueline L S; Subramaniam, Sriram (2009) Cryo-electron tomography of bacteria: progress, challenges and future prospects. Nat Rev Microbiol 7:666-75
Hirai, Teruhisa; Subramaniam, Sriram (2009) Protein conformational changes in the bacteriorhodopsin photocycle: comparison of findings from electron and X-ray crystallographic analyses. PLoS One 4:e5769
Khursigara, Cezar M; Wu, Xiongwu; Zhang, Peijun et al. (2008) Role of HAMP domains in chemotaxis signaling by bacterial chemoreceptors. Proc Natl Acad Sci U S A 105:16555-60
Khursigara, Cezar M; Wu, Xiongwu; Subramaniam, Sriram (2008) Chemoreceptors in Caulobacter crescentus: trimers of receptor dimers in a partially ordered hexagonally packed array. J Bacteriol 190:6805-10