Membrane proteins play critical roles in many cellular processes. High-resolution structures of membrane proteins provide keys to understand the mechanism of their functions. However, it is particularly difficult and challenging to determine high-resolution structure of membrane protein. One of the critical barriers in X-ray crystallography, in addition to membrane protein expression and purification, is at the step of crystallization. In this proposal, we will develop an innovative technology based on single particle electron cryo-microscopy (cryoEM) to determine structure of membrane proteins to subnanometer or higher resolution. In this method, membrane proteins are kept in their native conformation within lipid environment without need to form crystals. Single particle cryoEM has become a versatile tool in studying structures of soluble protein complexes without need of a large amount of proteins and forming crystals. It has achieved atomic resolution in studying viruses with icosahedral symmetry and near atomic resolution of well-behaved large protein complexes without high symmetry. However, applying this method to determine membrane protein structures to similar resolution is not straightforward. It requires innovative approach to overcome many technical difficulties, such as how to image very small membrane proteins in cryoEM and how to computationally align very noisy images of membrane proteins accurately. We proposed a novel approach to overcome these difficulties and to enable high-resolution structure determination of membrane proteins by single particle cryoEM. To test our novel approach, we will determine structures of two important membrane proteins: a bacterial homologue of mammalian vesicular glutamate transport (VGLUTs) and a Chloroquine resistance transporter (PfCRT). The VGLUTs transport glutamate into synaptic vesicle for regulated release by exocytosis, thus play a fundamental role in excitatory neurotransmission. The PfCRT plays a critical role in Plasmodium falciparum resistance to the quinoline antimalarials.
We aim to determine the structure of these two proteins to subnanometer resolution and eventually to near atomic resolution. We will also streamline our approach so that it can be applied to many other membrane proteins.
Membrane proteins play critical roles in many cellular and physiological processes. They are vital to human health and are drug targets for treatment of many human diseases. Structures of membrane proteins provide keys to understand the mechanism of their functions. Therefore, development of innovative technology to accelerate structure determination of membrane protein is of great biomedical importance.
| Palovcak, Eugene; Wang, Feng; Zheng, Shawn Q et al. (2018) A simple and robust procedure for preparing graphene-oxide cryo-EM grids. J Struct Biol 204:80-84 |
| Nguyen, Nam X; Armache, Jean-Paul; Lee, Changkeun et al. (2018) Cryo-EM structure of a fungal mitochondrial calcium uniporter. Nature 559:570-574 |
| Cormier, Anthony; Campbell, Melody G; Ito, Saburo et al. (2018) Cryo-EM structure of the ?v?8 integrin reveals a mechanism for stabilizing integrin extension. Nat Struct Mol Biol 25:698-704 |
| Kintzer, Alexander F; Green, Evan M; Dominik, Pawel K et al. (2018) Structural basis for activation of voltage sensor domains in an ion channel TPC1. Proc Natl Acad Sci U S A 115:E9095-E9104 |
| Autzen, Henriette E; Myasnikov, Alexander G; Campbell, Melody G et al. (2018) Structure of the human TRPM4 ion channel in a lipid nanodisc. Science 359:228-232 |
| Zhang, Yunxiao; Bulkley, David P; Xin, Yao et al. (2018) Structural Basis for Cholesterol Transport-like Activity of the Hedgehog Receptor Patched. Cell 175:1352-1364.e14 |
| Cheng, Yifan (2018) Single-particle cryo-EM-How did it get here and where will it go. Science 361:876-880 |
| Cheng, Yifan (2018) Membrane protein structural biology in the era of single particle cryo-EM. Curr Opin Struct Biol 52:58-63 |
| Dang, Shangyu; Feng, Shengjie; Tien, Jason et al. (2017) Cryo-EM structures of the TMEM16A calcium-activated chloride channel. Nature 552:426-429 |
| Jin, Peng; Bulkley, David; Guo, Yanmeng et al. (2017) Electron cryo-microscopy structure of the mechanotransduction channel NOMPC. Nature 547:118-122 |
Showing the most recent 10 out of 34 publications
