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.
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.
|Frank, Joachim (2017) The translation elongation cycle-capturing multiple states by cryo-electron microscopy. Philos Trans R Soc Lond B Biol Sci 372:|
|Feng, Xiangsong; Fu, Ziao; Kaledhonkar, Sandip et al. (2017) A Fast and Effective Microfluidic Spraying-Plunging Method for High-Resolution Single-Particle Cryo-EM. Structure 25:663-670.e3|
|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|
|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|
|Fu, Ziao; Kaledhonkar, Sandip; Borg, Anneli et al. (2016) Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy. Structure 24:2092-2101|
|Thompson, Colin D Kinz; 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 119:10888-10901|
|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|
|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:|
Showing the most recent 10 out of 100 publications