I was able to complete the cryo-EM aspects of WP2 and WP3. However, since nowadays structures obtained using cryo-EM are able to achieve resolutions comparable to X-ray crystallography, the crystallography aspects of WP2 and WP3 were not pursued. Instead I invested time in optimizing cryo-EM conditions for data collection (WP3) and processing (WP4). I collected two datasets on Titan Krios and one dataset on Polara, for the TFIIIC holocomplex with DNA, with direct-electron detectors. But likely due to inherent flexibility in the sample they did not yield high resolution. Hence, the WP4 has not yet been completed. In total, as evident from the above mentioned results I have made considerable progress towards attaining the cryo-EM structure of TFIIIC.
Alongside I studied the complete pre-initiation complex (PIC) comprising of TFIIIC, Brf1-TBP component of TFIIIB, RNA Polymerase III (Pol III) and DNA. With the minimal PIC now characterized (Vorländer et al, 2018), studying TFIIIC interactions with Pol III has been made feasible.
In total, I have studied the TFIIIC holocomplex for cryo-EM studies and with some preliminary experiments I have also explored the TFIIIC holocomplex structure in the bigger context of a transcription initiation complex. With ‘TF3C_EM’ I reinforced my ability to develop my own projects, work independently with a good balance in leadership and team-spirit.
During the time of the ‘TF3C_EM’ fellowship, I disseminated my results amongst the peers at international conferences - ‘Conference on methods and applications in the frontier between MX and CryoEM’ in 2017 in Barcelona, Spain being one of them. Here I discussed the technical aspects of my project with pioneers in the field of cryo-EM and modelling in X-ray crystallography. I also qualified for attending ‘The Lindau Noble Laureate meeting 2018’ (LiNo18), Lindau, Germany which was an ecstatic mix of people from various fields in biology. This ‘one-week’ long meeting was an amazing platform to discuss research with Nobel laureates and young scientists. The attendees came from various fields including physics, chemistry, biology and physiology. I was nominated as a Marie-Curie fellow and also had the opportunity to network with other fellows at the LiNo18.
I also attended the ‘Workshop on advanced topics in EM structure determination: challenges and opportunities’ organized by NRAMM in 2017 at the New York Structural Biology Center. There, I was able to build upon my cryo-EM expertise and network with some of the bright minds in the field. ‘TF3C_EM’ has given me the opportunity to not only build on my scientific expertise but also on my science communication skills with the above mentioned conferences and on-site courses at EMBL. This entire skill set is highly relevant for my future endeavors as an independent researcher.
