The long-term objective of this application is to develop methods for the efficient determination of atomic- resolution three-dimensional (3D) structures of large biological complexes in their native, non-crystalline states. The emerging technology of cryo-electron microscopy and 3D reconstruction (collectively "cryoEM") offers great promise for such structural studies. In the current funding period, the PI's group has developed data processing methods, implemented software, and validated these advances by determination of cryoEM structures of a number of large complexes to near-atomic resolution. We hypothesize that improvements in cryoEM technique, coupled with powerful computational tools, can be developed to create and process terabytes of image data for determination of atomic models of large complexes. Whereas we aimed and succeeded in the current funding period to obtain cryoEM structures at near-atomic resolution, the overall goal of this renewal application is to obtain structures at atomic resolution. In the renewal, we propose to improve both imaging itself and computation to bring resolution to 2.5E, permitting identification of amino acids and ultimately atomic resolution. In the renewal period, we therefore focus on improvement of imaging and methods for image acquisition and correction (Aim #1), making cryoEM structure determination hundreds of times more efficient and affordable (Aim #2), extending our success with icosahedral objects to helical ones (Aim #3), improvement of cryoEM-specific atomic-model refinement software that takes advantage of phase as well as amplitude data and that can handle the vastly greater amounts of data required for atomic resolution 3D reconstruction (Aim #4), and validation by applying these new methods for atomic structure determination to a small icosahedral hepatitis B virus (HBV) core, a large icosahedral aquareovirus and the helical tobacco mosaic virus (TMV) (Aim #5). Our overriding principle in achievement of all of these aims is that the optimized methods we create should permit them to be used routinely, reproducibly, and affordably. A successful outcome of this renewal project will remove the final obstacles towards determination of atomic- resolution structures for large complexes by cryoEM and will have great impact on many areas of biomedical research. This achievement will complement other structural methods, particularly X-ray crystallography of purified proteins that are amenable to crystallization and NMR of small molecules in solution. Specifically, this achievement will enable investigators to place individual proteins within the structural context of the larger assembly and to visualize native shapes and physical chemical interactions among the parts of the complex. Moreover, the ability to look at large complexes at atomic resolution will permit visualization of complexes bound to antibodies, receptors, and drugs.

Public Health Relevance

This continuation project builds on the current success in reaching near-atomic resolution cryo-electron microscopy and aims to make atomic resolution structure determination a routine practice for large biological complexes. Realization of this goal will have far-reaching impact on multiple biomedical areas including structural biology, biochemistry, cell biology, virology, pathology, and molecular medicine. A successful outcome of this project will create a new paradigm for the entire biological structure community. Atomic- resolution cryoEM would also serve the mission of NIH by speeding up structural studies of disease mechanisms and by generating atomic information for rational drug design.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM071940-09
Application #
8501521
Study Section
Microscopic Imaging Study Section (MI)
Program Officer
Flicker, Paula F
Project Start
2006-05-01
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
9
Fiscal Year
2013
Total Cost
$279,472
Indirect Cost
$95,036
Name
University of California Los Angeles
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Buehler, Daniel C; Marsden, Matthew D; Shen, Sean et al. (2014) Bioengineered vaults: self-assembling protein shell-lipophilic core nanoparticles for drug delivery. ACS Nano 8:7723-32
Zhou, Z Hong; Hui, Wong Hoi; Shah, Sanket et al. (2014) Four levels of hierarchical organization, including noncovalent chainmail, brace the mature tumor herpesvirus capsid against pressurization. Structure 22:1385-98
Gurel, Pinar S; Ge, Peng; Grintsevich, Elena E et al. (2014) INF2-mediated severing through actin filament encirclement and disruption. Curr Biol 24:156-64
Ge, Peng; Zhou, Z Hong (2014) Chaperone fusion proteins aid entropy-driven maturation of class II viral fusion proteins. Trends Microbiol 22:100-6
Zhou, Z Hong (2014) Structures of viral membrane proteins by high-resolution cryoEM. Curr Opin Virol 5:111-9
Dai, Xinghong; Gong, Danyang; Wu, Ting-Ting et al. (2014) Organization of capsid-associated tegument components in Kaposi's sarcoma-associated herpesvirus. J Virol 88:12694-702
Jiang, Jiansen; Magilnick, Nathaniel; Tsirulnikov, Kirill et al. (2013) Single particle electron microscopy analysis of the bovine anion exchanger 1 reveals a flexible linker connecting the cytoplasmic and membrane domains. PLoS One 8:e55408
Slaymaker, Ian M; Fu, Yang; Toso, Daniel B et al. (2013) Mini-chromosome maintenance complexes form a filament to remodel DNA structure and topology. Nucleic Acids Res 41:3446-56
Yu, Xuekui; Jin, Lei; Jih, Jonathan et al. (2013) 3.5A cryoEM structure of hepatitis B virus core assembled from full-length core protein. PLoS One 8:e69729
Zhang, Xiaokang; Ge, Peng; Yu, Xuekui et al. (2013) Cryo-EM structure of the mature dengue virus at 3.5-A resolution. Nat Struct Mol Biol 20:105-10

Showing the most recent 10 out of 47 publications