This proposal seeks continued support for exploration of structure and function of the ribosome actively engaged in protein synthesis, by cryo-electron microscopy (cryo-EM) and single-particle reconstruction. Protein biosynthesis is one of the most fundamental processes of life, but despite seminal work in solving the structure of the ribosome, this process is not well understood. Cryo-EM is uniquely suited to capture the molecule in functional states under close to native conditions. In this lab, several seminal discoveries regarding the dynamics of this process in both eubacterial and eukaryotic translation have been made in the past;among these the ratchet-like motion during mRNA-tRNA translocation and the large spring-like deformation of the aminoacyl-tRNA as it enters the ribosome during the aminoacyl-tRNA selection process. Special focus of this research program will be the eukaryotic (yeast and mammalian) ribosome.
The aims of the proposed studies are threefold: (1) to make use of tilt pair recoding, novel technology for online data capture in the electron microscope, workflow optimization, and availability of a Titan Krios instrument at Janelia Farm, and other improvements in order to achieve atomic (better than 3.5A) resolution for selected ribosome complexes of special interest, such as ribosomes of trypanosomes and a yeast translocation complex;(2) to study the process of mammalian eukaryotic translation initiation by cryo-EM visualization of selected initiation complexes;(3) to study, with the aid of advanced classification methods, the distribution of states and their structural manifestations in a freely equilibrating, factor-free, eukaryotic ribosome sample as a function of relevant parameters such as temperature and ionic conditions. Samples will be obtained from several collaborating labs that are specialized in eukaryotic translation. Additional important collaborations are in the area of eukaryotic ribosome structure, smFRET, classification of heterogeneous samples by manifold embedding, and atomic modeling. Density maps when obtained at sufficient resolution will be analyzed by flexible fitting and interpreted in the rich context of existing structural, kinetics, single-molecule FRET, and biochemical data.

Public Health Relevance

The ribosome performs protein synthesis in all cells in all life forms. The research proposed will further the understanding of its functional dynamics, and thus contribute to a fundamental understanding of all life processes. Understanding the function of the eukaryotic ribosome on a fundamental level is advancing our ability to combat debilitating human diseases arising from dysfunctions of translation, and viral takeover of the ribosome by the IRES mechanism (e.g., in hepatitis C). This knowledge also helps in developing drugs to fight eukaryotic parasites.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029169-32
Application #
8666646
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Flicker, Paula F
Project Start
1982-04-01
Project End
2016-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
32
Fiscal Year
2014
Total Cost
$477,463
Indirect Cost
$174,842
Name
Columbia University (N.Y.)
Department
Biochemistry
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
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Frank, Joachim (2017) Time-resolved cryo-electron microscopy: Recent progress. J Struct Biol 200:303-306
Liu, Zheng; Gutierrez-Vargas, Cristina; Wei, Jia et al. (2017) Determination of the ribosome structure to a resolution of 2.5 Å by single-particle cryo-EM. Protein Sci 26:82-92
Twomey, Edward C; Yelshanskaya, Maria V; Grassucci, Robert A et al. (2017) Channel opening and gating mechanism in AMPA-subtype glutamate receptors. Nature 549:60-65
Malyutin, Andrey G; Musalgaonkar, Sharmishtha; Patchett, Stephanie et al. (2017) Nmd3 is a structural mimic of eIF5A, and activates the cpGTPase Lsg1 during 60S ribosome biogenesis. EMBO J 36:854-868
Frank, Joachim (2017) The translation elongation cycle-capturing multiple states by cryo-electron microscopy. Philos Trans R Soc Lond B Biol Sci 372:
Twomey, Edward C; Yelshanskaya, Maria V; Grassucci, Robert A et al. (2017) Structural Bases of Desensitization in AMPA Receptor-Auxiliary Subunit Complexes. Neuron 94:569-580.e5
Frank, Joachim (2017) Advances in the field of single-particle cryo-electron microscopy over the last decade. Nat Protoc 12:209-212
Maji, Suvrajit; Shahoei, Rezvan; Schulten, Klaus et al. (2017) Quantitative Characterization of Domain Motions in Molecular Machines. J Phys Chem B 121:3747-3756
Frank, Joachim (2016) Whither Ribosome Structure and Dynamics Research? (A Perspective). J Mol Biol 428:3565-9

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