In the past week, we have been able to find a resemblance of EF-G at the 560-millisecond time point using time-resolved cryo-EM. The elongation factor that we captured does not 100% resemble EF-G, but there are similarities to it. Therefore, it is predicted that it is a combination of both EF-Tu and EF-G. The resulting structural map is colored for ease of understanding. The 50S subunit (Blue) and the 30S subunit (Yellow) make up the entirety of the ribosome, a 70S structure. The green structure in the map represents a tRNA. In the future, we must continue to test multiple time points in effort to catch EF-G at the highest resolution.
As I mentioned in my previous report, I was also working on ribosome purification. After repeating the entire process, three times, since we did not get any results the first two times - which took about a month and a half - we finally decided to work in the lab that published the protocol we were following. After learning about the techniques, a little more, we were able to extract approximately 8 mL of purified ribosomes from a 6-liter culture. The next step is to prepare grids using the sample and examine the quality of the ribosomes. This experience has taught me that research requires perseverance and patience. Although one may be following an exact protocol, small things may affect the results. I also learned that there is no timeline for research, since small discoveries can be made along the way.
The past 10 weeks working in the Frank lab has been very enlightening, to say the least. I have been able to learn techniques in a new field of biology, which has helped widen my knowledge of science. For instance, I learned how to use electron microscopes efficiently and how to collect data using them. This was very different from using a light or phase microscope and manually counting cells. Through this 10-week experience, I have also learned how to code and conduct data processing. In addition, I was also able to visit New York Structural Biology Center and use their Krios microscope, which is much larger than the microscopes we have in the Frank lab for data collection. Of all the techniques I have learned, I thought the most interesting techniques to learn were the blotting and spraying techniques. Due to their intricacies, it was hard to use the techniques independently at first. But, after practice and numerous trials, the techniques became easier to independently manage. It was fulfilling to use the blotting method, pinpoint an issue and then use an alternate technique to solve the issue.
At the Frank lab, there are many people that specialize in different aspects of grid preparation, data collection and data processing and there are a few that do all three themselves. The lab is fundamentally based on team work and it has been great to become part of the Frank lab team. I was pushed to think independently and find solutions for the problems we encountered, as well as collaborating with others to solve the issues. Furthermore, I will be continuing collecting data on EF-G in the Frank lab, during the academic year, in effort to capture a high-resolution structure.
In summary, this research experience has been extremely fruitful as it has made me understand my passion for research. I never considered pursuing research along with medicine, but this internship has opened my eyes to the numerous opportunities that are available in the STEM field. I hope to embark upon a career that allows to pursue both my passions, research and medicine. I have also been working rigorously on completing my research poster, which I will be presenting on July 29th at the Barnard SRI symposium. I look forward to presenting all the work I have been doing, during these 10 weeks, as well as, learning about what my peers have been doing this summer.