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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM098672-04
Application #
8706184
Study Section
Special Emphasis Panel (ZGM1)
Program Officer
Chin, Jean
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Wang, Ray Yu-Ruei; Song, Yifan; Barad, Benjamin A et al. (2016) Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta. Elife 5:
Frauenfeld, Jens; Löving, Robin; Armache, Jean-Paul et al. (2016) A saposin-lipoprotein nanoparticle system for membrane proteins. Nat Methods 13:345-51
Gao, Yuan; Cao, Erhu; Julius, David et al. (2016) TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action. Nature 534:347-51
Wu, Shenping; Armache, Jean-Paul; Cheng, Yifan (2016) Single-particle cryo-EM data acquisition by using direct electron detection camera. Microscopy (Oxf) 65:35-41
Zhao, Minglei; Wu, Shenping; Zhou, Qiangjun et al. (2015) Mechanistic insights into the recycling machine of the SNARE complex. Nature 518:61-7
Barad, Benjamin A; Echols, Nathaniel; Wang, Ray Yu-Ruei et al. (2015) EMRinger: side chain-directed model and map validation for 3D cryo-electron microscopy. Nat Methods 12:943-6
Wang, Ray Yu-Ruei; Kudryashev, Mikhail; Li, Xueming et al. (2015) De novo protein structure determination from near-atomic-resolution cryo-EM maps. Nat Methods 12:335-8
DiMaio, Frank; Song, Yifan; Li, Xueming et al. (2015) Atomic-accuracy models from 4.5-Ã… cryo-electron microscopy data with density-guided iterative local refinement. Nat Methods 12:361-5
Paulsen, Candice E; Armache, Jean-Paul; Gao, Yuan et al. (2015) Structure of the TRPA1 ion channel suggests regulatory mechanisms. Nature 520:511-7
Kim, JungMin; Wu, Shenping; Tomasiak, Thomas M et al. (2015) Subnanometre-resolution electron cryomicroscopy structure of a heterodimeric ABC exporter. Nature 517:396-400

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