This proposal seeks continued support for exploration of structure and function of the ribosome actively engaged in protein synthesis, by cryo-electron microscopy and single-particle reconstruction. Several seminal discoveries regarding the dynamics of this process have been made in pursuit of this approach;among these the ratchet motion during mRNA-tRNA translocation and the spring-like deformation of the aminoacyl-tRNA as it enters the ribosome. The emphasis of the proposed studies is twofold: to characterize suitably stabilized transition states during both decoding and translocation at high resolution, and to study the time course of both processes using time-resolved cryo-electron microscopy (ms range) of pre-equilibrium samples. A novel monolithic microfluidic mixing and spraying device developed at the NCRR/NIH RVBC Resource at the Wadsworth Center in Albany will be tested and used in collaboration with the Resource, and the technology will be later transferred to the Columbia lab. Samples will be obtained from collaborators Drs. Rachel Green, Johns Hopkins University and Mans Ehrenberg, Uppsala University. Additional collaborations are in the areas of classification of heterogeneous samples and Molecular Dynamics simulations. Density maps when obtained at sufficient resolution will be analyzed by flexible fitting and interpreted in the rich context of structural, kinetics, single-molecule FRET, and biochemical data.

Public Health Relevance

The ribosome performs protein synthesis in all cells. The research proposed will further the understanding of its functional dynamics, and thus contribute to a fundamental understanding of all life processes. Specifically, understanding the function of bacterial ribosomes is furthering our ability to combat drug resistance.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM055440-17
Application #
8501509
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Flicker, Paula F
Project Start
1997-01-01
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
17
Fiscal Year
2013
Total Cost
$304,919
Indirect Cost
$113,849
Name
Columbia University (N.Y.)
Department
Biochemistry
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
Fu, Ziao; Kaledhonkar, Sandip; Borg, Anneli et al. (2016) Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy. Structure 24:2092-2101
Liu, Zheng; Gutierrez-Vargas, Cristina; Wei, Jia et al. (2016) Determination of the ribosome structure to a resolution of 2.5 Ã… by single-particle cryo-EM. Protein Sci :
Chen, Bo; Frank, Joachim (2016) Two promising future developments of cryo-EM: capturing short-lived states and mapping a continuum of states of a macromolecule. Microscopy (Oxf) 65:69-79
Frank, Joachim; Ourmazd, Abbas (2016) Continuous changes in structure mapped by manifold embedding of single-particle data in cryo-EM. Methods 100:61-7
Li, Wen; Liu, Zheng; Koripella, Ravi Kiran et al. (2015) Activation of GTP hydrolysis in mRNA-tRNA translocation by elongation factor G. Sci Adv 1:
Kinz-Thompson, Colin D; Sharma, Ajeet K; Frank, Joachim et al. (2015) Quantitative Connection between Ensemble Thermodynamics and Single-Molecule Kinetics: A Case Study Using Cryogenic Electron Microscopy and Single-Molecule Fluorescence Resonance Energy Transfer Investigations of the Ribosome. J Phys Chem B 119:10888-901
Chen, Bo; Kaledhonkar, Sandip; Sun, Ming et al. (2015) Structural dynamics of ribosome subunit association studied by mixing-spraying time-resolved cryogenic electron microscopy. Structure 23:1097-105
Chen, Bo; Shen, Bingxin; Frank, Joachim (2014) Particle migration analysis in iterative classification of cryo-EM single-particle data. J Struct Biol 188:267-73
Chen, Bo; Boël, Grégory; Hashem, Yaser et al. (2014) EttA regulates translation by binding the ribosomal E site and restricting ribosome-tRNA dynamics. Nat Struct Mol Biol 21:152-9
Dashti, Ali; Schwander, Peter; Langlois, Robert et al. (2014) Trajectories of the ribosome as a Brownian nanomachine. Proc Natl Acad Sci U S A 111:17492-7

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