This summer, I am conducting research in the Frank lab at Columbia University Medical Center under the mentorship of Dr. Joachim Frank. I initially started working at the Frank Lab last semester; however, working full time during the summer has been a completely different experience. I am able to work on multiple projects with more commitment. The lab mainly focuses on protein and ribosome reconstruction using cryo-electron microscopy. Cryo-EM is a technique used to study samples at liquid nitrogen temperatures to achieve molecular resolution. It allows researchers to understand in vivo functions using in vitro techniques.
Currently, I am studying the conformation of Elongation Factor-G (EF-G) at various time intervals - during the elongation process of proteins - in efforts to visualize all the steps of the elongation cycle. I am observing various time intervals by changing the time for the elongation reaction. However, when it came to setting up the grids that were used to analyze the sample using cryo-EM, we were expected to have the sample on the grid, and immediately plunged into liquid nitrogen within 5-10 seconds. While performing this step, I realized that it was difficult to control this time, and that we must find a different method, in order to keep the time consistent. Therefore, we are simultaneously working on another project that essentially will control the time it takes for the sample to be placed on the grid; it will optimize the spread/resolution of the sample. As of now, we have been testing this time-resolved technique with Apoferritin, a homogeneous protein, but the goal is to use this technique to analyze EF-G at different time points more accurately and efficiently.
I have learned not only how to successfully load a sample onto a grid and the grid onto an electron microscope, but also, I learned coding and data processing. Loading a sample onto a grid for electron microscopy is more complicated and precise than preparing a slide to observe under a light microscope. To prepare an cryo-EM grid, the grid needs to be coated with carbon, plasma cleaned, and plunged into liquid nitrogen. The grid must be handled with tweezers throughout the entire process, which is difficult as the grid is susceptible to carbon breakage, since the grid is very small and flimsy. In addition, loading the grid into the holder to insert into the electron microscope is also tricky as the grid can be damaged, if not performed correctly. All these steps require one’s acute attention and precision. Moreover, collecting and analyzing data using an electron microscope is all computerized. I learned how to start the microscope, insert the cryo-holder into the microscope and collect data properly. After data collection, the data must be processed, which required me to learn how to code using Linux. At first, this was very intimidating, as I had no previous knowledge of coding, but after observing and practicing code, I was able to pick it up.
My goals for the Frank lab are to learn about another aspect of the Biology field and learn techniques that I have not learned before. I had done work with mammalian tissue cultures and light/plasma microscopes. Therefore, the work I am currently doing is very different; it gives me more perspective of the different opportunities of work available as a researcher. I am hoping to continue learning new techniques in the lab and perfecting the old ones. I look forward to, hopefully, having success with the EF-G and time-resolved experiments, both of which are novel ideas.