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 #
5R01GM071940-08
Application #
8300856
Study Section
Microscopic Imaging Study Section (MI)
Program Officer
Flicker, Paula F
Project Start
2006-05-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
8
Fiscal Year
2012
Total Cost
$289,608
Indirect Cost
$98,483
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
Zhang, Xing; Lai, Mason; Chang, Winston et al. (2016) Structures and stabilization of kinetoplastid-specific split rRNAs revealed by comparing leishmanial and human ribosomes. Nat Commun 7:13223
Toso, Daniel B; Javed, Muhammad Mohsin; Czornyj, Elizabeth et al. (2016) Discovery and Characterization of Iron Sulfide and Polyphosphate Bodies Coexisting in Archaeoglobus fulgidus Cells. Archaea 2016:4706532
Jurado, Ashley R; Huang, Christine S; Zhang, Xing et al. (2015) Structure and substrate selectivity of the 750-kDa α6β6 holoenzyme of geranyl-CoA carboxylase. Nat Commun 6:8986
Dai, Xinghong; Gong, Danyang; Xiao, Yuchen et al. (2015) CryoEM and mutagenesis reveal that the smallest capsid protein cements and stabilizes Kaposi's sarcoma-associated herpesvirus capsid. Proc Natl Acad Sci U S A 112:E649-56
Zhang, Xing; Ding, Ke; Yu, Xuekui et al. (2015) In situ structures of the segmented genome and RNA polymerase complex inside a dsRNA virus. Nature 527:531-4
Jiang, Jiansen; Chan, Henry; Cash, Darian D et al. (2015) Structure of Tetrahymena telomerase reveals previously unknown subunits, functions, and interactions. Science 350:aab4070
Ge, Peng; Scholl, Dean; Leiman, Petr G et al. (2015) Atomic structures of a bactericidal contractile nanotube in its pre- and postcontraction states. Nat Struct Mol Biol 22:377-82
Clemens, Daniel L; Ge, Peng; Lee, Bai-Yu et al. (2015) Atomic structure of T6SS reveals interlaced array essential to function. Cell 160:940-51
Yu, Xuekui; Jiang, Jiansen; Sun, Jingchen et al. (2015) A putative ATPase mediates RNA transcription and capping in a dsRNA virus. Elife 4:e07901
Volland, Stefanie; Hughes, Louise C; Kong, Christina et al. (2015) Three-dimensional organization of nascent rod outer segment disk membranes. Proc Natl Acad Sci U S A 112:14870-5

Showing the most recent 10 out of 68 publications