Scientists at the Bengaluru-based Institute of Stem Cell Biology and Regenerative Medicine (InStem) and National Centre for Biological Sciences (NCBS) have unravelled the structure of a bacterial enzyme, PaaZ, using cryo-electron microscopy. The study was conducted in collaboration with the MRC Laboratory of Molecular Biology, Cambridge, UK and Carver College of Medicine, University of Iowa, USA. The structure was recently published in the journal Nature Communications.
This is the latest high resolution structure (less than 3A) to be published from the recently established state-of-the-art single electron Cryo-EM facility at Bangalore Life Science Cluster.
Here is an exclusive interview with Nitish Sathyanarayanan, one of the first authors of the paper, on this incredible journey.
1. What got you interested in structural biology?
I began to get fascinated by structural biology during my Bachelor’s degree, where I spent a lot of time reading Lehninger “Principles of Biochemistry”. If you notice keenly, almost every section of this bible contains a structural explanation. It was this interest in biochemistry and enzymology that got me into the field of structural biology. Since I come from Biology training (not Physics or Chemistry), I have always used structural biology as a set of ‘tools/techniques’ to understand the functions of enzymes.
2. How did you come to study the structure of PaaZ?
We stumbled upon PaaZ due to our interest in multi-domain proteins which are involved in aromatic ring degradation. Phenyl Acetate (Paa) degradation pathway is also called “hybrid pathway” since its mechanism of ring degradation has features of both aerobic and anaerobic pathways. Our specific interest was to understand the functioning of this enzyme through its structure and biochemistry. It is also important since several environmental pollutants such as styrene converge to Paa through peripheral pathways.
3. What were the challenges you faced in the project?
We were essentially trying to understand how the enzyme functioned using its structure. We spent nearly 18-24 months crystallizing the protein, with no success in obtaining a diffraction quality crystal. We then attempted an integrated structure modelling approach (Light scattering, SAXS, MD and Modelling) to obtain a model with limited success. This was also the time when structural biology domain was undergoing “resolution revolution” (the term used to describe new advances in Cryo-EM). Since the protein was big and we had limited success with other methods, we decided to explore Cryo-EM to understand the structure.
4. What was your reaction when you first solved these structures?
I was amazed! Here was a protein that I purified for several years. I had only imagined it as a liquid or a blue band on an SDS-PAGE gel or as a gel filtration profile. I never knew how it looked. It was like a blind date. When you talk to someone only over the phone for several years, you build your own imagination. It was similar. But when I saw the structure as a Cryo-EM map, it was more beautiful than I ever imagined.
5. How do you plan on taking the findings of this project further as a scientist and entrepreneur?
For me, PhD was “one big project” since I was involved with multiple projects and simultaneously part of three labs (Bioinformatics, Structural biology and Cryo-EM). As my mentor Rams (Prof. S. Ramaswamy) always said, PhD is training – training an individual to ask tough questions, design an experiment to answer these questions, perform and reproduce an experiment, and find a conclusion. In between these four steps, a PhD student often goes through innumerable failed experiments. It is the ability to break a complex problem into simpler tasks, find answers to these smaller tasks (with several failures) and, in the end, put all pieces of the puzzle to build a complete picture. This is a life lesson I plan to leverage in my next journey as a tech entrepreneur.
6. Your message to academic peers struggling in their PhD?
If you are not struggling, then there is a problem. It’s called “re-search” for a reason. I feel that one should have read at least about a hundred papers in that area of research in the first 6 months. My humble suggestion is to spend a considerable amount of time reading and staying updated with literature. This can be a great source of new ideas. The other benefit is that you will become aware of the labs that work in a similar field. You could always request for plasmids, protocols, reagents, suggestions etc. from them. If you do not read enough, you cannot do any of this. And please remember, by the end of your PhD, you should have become an expert in that field.
Picture adapted from the published paper.
Link to the full-text of the article: https://www.nature.com/articles/s41467-019-11931-1
Joel P Joseph 