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 by cryo electron microscopy (cryoEM). In the current funding period, the PI's group has developed data acquisition and processing methods, implemented software, and validated these advances by determination of cryoEM structures of a number of large icosahedral and helical complexes, as well as complexes of low or no symmetry, some to an unprecedented 2.9A resolution. We hypothesize that further advances in cryoEM and single-particle reconstruction can be obtained by taking advantage of electron counting and """"""""super-resolution"""""""" capabilities of direct electron detectors for determination of atomic models for even larger or transiently stable complexes. Whereas we aimed and succeeded in the current funding period to obtain cryoEM structures at atomic resolution for icosahedral and helical viruses, the goal of this renewal application is to develop methods for atomic structures for even larger complexes, including those that are only transiently stable. In the renewal, we propose to improve both imaging itself and computation to bring resolution to ~2A, permitting identification of small ligands or drugs in large complexes, even resolving holes of benzene rings of aromatic side chains. Specifically, we will develop novel imaging and image correction methods that take advantage of the practically noise-free images afforded by a newly installed direct electron detector (Aim #1);achieve high-resolution reconstructions of complexes that are deformable or intrinsically structurally heterogeneous by a symmetry- based selection method (Aim #2);test use of de novo atomic models to refine orientation and center parameters to further improve map resolution and model accuracy (Aim #3);and validate these new methods for atomic structure determination by application to the icosahedral complexes cytoplasmic polyhedrosis virus (CPV), herpesvirus capsids and membrane-containing dengue virus, filamentous complexes including tobacco mosaic virus (TMV), NS1 tubule and actin, and other biological machines such as pyocin, pyruvate dehydrogenase and telomerase complexes (Aim #4). A successful outcome of this renewal project will further push the resolution limit of cryoEM in structural studies of large complexes 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.

Public Health Relevance

This continuation project builds on the success in reaching the unprecedented resolution with cryoEM technology, even 2.9A for a dsRNA virus, and aims to make atomic resolution structure determination a routine practice for large biological and even transiently stable 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. Thanks to these advances, atomic-resolution cryoEM is now poised to contribute to unveiling mechanisms of human disease and to designing counter measures.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM071940-10
Application #
8762345
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Flicker, Paula F
Project Start
2006-05-01
Project End
2018-06-30
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
10
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Tian, Tian; Li, Xiaorun; Liu, Yingying et al. (2018) Molecular basis for CENP-N recognition of CENP-A nucleosome on the human kinetochore. Cell Res 28:374-378
Ding, Ke; Nguyen, Lisa; Zhou, Z Hong (2018) In Situ Structures of the Polymerase Complex and RNA Genome Show How Aquareovirus Transcription Machineries Respond to Uncoating. J Virol 92:
Ding, Ke; Zhang, Xing; Mrazek, Jan et al. (2018) Solution Structures of Engineered Vault Particles. Structure 26:619-626.e3
Huynh, Kevin W; Jiang, Jiansen; Abuladze, Natalia et al. (2018) CryoEM structure of the human SLC4A4 sodium-coupled acid-base transporter NBCe1. Nat Commun 9:900
Zhu, Rui; Xu, Longfa; Zheng, Qingbing et al. (2018) Discovery and structural characterization of a therapeutic antibody against coxsackievirus A10. Sci Adv 4:eaat7459
Park, Kyoungwon; Kuo, Yung; Shvadchak, Volodymyr et al. (2018) Membrane insertion of-and membrane potential sensing by-semiconductor voltage nanosensors: Feasibility demonstration. Sci Adv 4:e1601453
Jiang, Jiansen; Baiesc, Flavius L; Hiromasa, Yasuaki et al. (2018) Atomic Structure of the E2 Inner Core of Human Pyruvate Dehydrogenase Complex. Biochemistry 57:2325-2334
Dai, Xinghong; Gong, Danyang; Lim, Hanyoung et al. (2018) Structure and mutagenesis reveal essential capsid protein interactions for KSHV replication. Nature 553:521-525
Ho, Chi-Min; Beck, Josh R; Lai, Mason et al. (2018) Malaria parasite translocon structure and mechanism of effector export. Nature 561:70-75
Guenther, Elizabeth L; Ge, Peng; Trinh, Hamilton et al. (2018) Atomic-level evidence for packing and positional amyloid polymorphism by segment from TDP-43 RRM2. Nat Struct Mol Biol 25:311-319

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