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.
In my previous entry, I mentioned that we were analyzing the Apoferritin data to see if it was possible to achieve high resolution with time-resolve cryo-EM. It is very exciting to report that we were able to achieve a resolution of 3 Angstroms, which is the highest resolution yet! Since then, I have been working with time-resolve cryo-EM in effort to capture elongation factor G. When I was screening the grid, the first week, I saw an unusually large amount of contamination of ice on the grid and no droplets. However, after collecting data with Apoferritin, we expected to see a large number of drops and minimal contamination.
To pinpoint the source of the problem, we decided to test the polymix buffer, since it was the buffer the sample was dissolved in. However, polymix buffer is not the ideal buffer to use for time-resolved cryo-EM as it is very viscous so it will not spray as well as low salt PBS buffer: the buffer used with Apoferritin. The polymix buffer showed evidence of large amounts of contamination and round drops that did not spread. We hypothesized that the contamination could possibly be due to the salt build-up in the spray-nozzle. Between every grid preparation, we started to clean the nozzle with water. In addition, some of the holes had ice that resembled the texture of snakeskin; the ice should normally appear to be very smooth. We figured that the texture of the ice could be solved by checking the temperature of the cryogen - a mixture of one-part ethane and two-parts propane that the grid is plunged into after the sample is dispensed on it - before each grid is prepared; the temperature should ideally be under -150° Celsius. To make the drops spread more, the girds were plasma cleaned for longer time to make them more hydrophilic. After fixing these issues, the resulting grids looked very clean.
After the issues with the polymix buffer were resolved, we prepared grids with the EF-G sample. During data collection, we found many droplets and minimal contamination at the time point of 560 milliseconds. We found that the optimal place to collect data with the most particles with ribosomes was near the grid bar, which is near the edge of the square, since the grid bar would absorb some sample and help the drop spread. Data collection resulted in over 80,000 particles. These particles underwent data processing in which particles were picked, classified, and refined. Finally, we had a 3D map of the ribosome. This map demonstrated a resemblance of an elongation factor, but the resolution was not high enough for us to conclude that it is definitely EF-G. There are two types of elongation factors, EF-G and EF-Tu. The purpose of this experiment is to observe EF-G, however, the map had a resemblance of both elongation factors. We have to continue to prepare grids at different time points and increase the concentration of EF-G in hopes to capture a high-resolution image of EF-G.
During the past two weeks, I have been, in parallel, learning about ribosome purification. We are trying to extract ribosomes from bacterial E. coli cultures to study initiation factor 2 using time-resolved cryo-EM. Regardless of the many set-backs such as a broken centrifuge, we were able to complete the entire process once. Although we were not able to see many peaks which meant we did not have polysomes, 70S ribosomes, 50S subunits or 30S subunits, we became accustomed to the process of ribosome purification. We are currently repeating the entire process, in hopes, to finally purify ribosomes that can be analyzed.
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.