Title: Time-resolved Cryo-EM Studies on Translation and Cryo-EM Studies on Membrane Proteins
Description: Single-particle reconstruction technique is one of the major approaches to studying ribosome structure and membrane proteins. In this thesis, I report the use of time-resolved cryo-EM technique to study the structure of short-lived ribosome complexes and conventional cryo-EM technique to study the structure of ribosome complexes and membrane proteins. The thesis consists three parts. The first part is the development of time-resolved cryo-EM technique. I document the protocol for how to capture short-lived states of the molecules with time-resolved cryo-EM technique using microfluidic chip. Working closely with Dr. Lin’s lab at Columbia University Engineering Department, I designed and tested a well-controlled and effective microspraying-plunging method to prepare cryo-grids. I demonstrated the performance of this device by a 3-Å reconstruction from about 4000 particles collected on grids sprayed with apoferritin suspension. The second part is the application of time-resolved cryo-EM technique for studying short-lived ribosome complexes in bacteria translation processes on the time-scale of 10-1000 ms.

I document three applications on bacterial translation processes. The initiation project is collaborated with Dr. Gonzalez’s lab at Chemistry Department, Columbia University. The termination and recycling projects are collaborated with Dr. Ehrenberg’s lab at Department of Cell and Molecular Biology, Uppsala University. I captured and solved short-lived ribosome intermediates complexes in these processes. The results demonstrate the power of time-resolved cryo-EM to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology. The last part is the application of conventional cryo-EM technique to study ribosome complexes and membrane proteins. This part includes five collaboration projects. Human GABA(B) receptor project is the collaboration with Dr. Fan at Department of Pharmacology, Columbia University. Cyclic nucleotide-gated (CNG) channels project is the collaboration with Dr. Yang at Department of Biological Sciences, Columbia University.

The cryo-EM study of Ybit-70S ribosome complex and Cystic fibrosis transmembrane conductance regulator (CFTR) project are the collaboration with Dr. Hunt at Department of Biological Sciences, Columbia University. The cryo-EM study of native lipid bilayer in membrane protein transporter is the collaboration with Dr. Hendrickson at Department of Biochemistry and Molecular Biophysics, Columbia University and Dr. Guo at Department of Medicinal Chemistry, Virginia Commonwealth University.