Single Particle Short Course
March 2-6, 2020
On the first week of March, NCCAT held its first ever week-long short course on the theory and practice of single-particle analysis. This also happened to coincide with the first confirmed coronavirus case in New York City, so it ended up being the last gathering any of us would have for some months. Eighteen students from all over the country (and a few outside) attended lectures from leaders in the field, including Joachim Frank, Fred Sigworth, and Tom Walz were combined with hands on exercises for the full SPA pipeline, from grid prep to model building.
Three of the students agreed to answer a few questions about themselves, their work, and their experience taking the course for this newsletter. Sohail Khoshnevis, from Emory was one of the students who brought his own sample to collect data on during the course; Irena Bregy, one of the international students who visited from Switzerland; and Emila Arturo, no stranger to New York City but currently works in La Jolla, California.*
Tell us about yourself and what you do.
Emilia Arturo: My name is Emilia Arturo, but I often go by ‘Emily’. I am a post-doctoral associate in the laboratory of Erica Ollmann Saphire at the La Jolla Institute for Immunology in the Division of Structural Biology & Infectious Diseases, which is located in La Jolla, California. I was born in Romania, and since coming to the US as a child I’ve lived in 8 states.
Irina Bregy: I’m from a small village in the south of Switzerland. For my studies (Bachelor in Cell Biology, Master in Molecular Biology), I moved to Bern (Switzerland). Bern is my home for almost 6 years now. With the start of my PhD (one year ago), I dove into the field of structural biology.
Sohail Khoshnevis: My name is Sohail Khoshnevis and I am a scientist at Emory University in Atlanta.
What are you working on now?
EA: I am most involved in a project related to investigating the structure and antigenicity of the single surface glycoprotein of Machupo virus (MACV). MACV is an arenavirus that is endemic to South America, Bolivia in particular. It is the cause of a hemorrhagic fever similar to Lassa fever. MACV passes from humans to humans and is ~25% lethal. Despite it having been discovered about 60 years ago, there is still no vaccine or treatment that is highly effective against this virus. Our main tools for studying its surface glycoprotein, as well as the antibodies that bind and neutralize it, are X-ray crystallography and cryo-electron microscopy. Our goal is to use structural biology to develop an appropriate antigen based on the MACV surface glycoprotein for vaccination and antibody discovery.
IB: I am working with Trypanosoma brucei. T. brucei is a single celled eukaryotic parasite causing Human African Trypanosomiasis, and the cattle’s disease Nagana. Using cryo-electron tomography, and single particle cryoEM, I am investigating the mitochondrial genome segregation machinery of the parasite.
SK: I study eukaryotic protein synthesis and different complexes which regulate this process.
Why did this type of work interest you and how did you get started?
EA: I wished to work with viral proteins because they are notoriously challenging to study in isolation. The membrane embedded glycoproteins are particularly challenging in their native form, so require clever engineering to make them amenable to our structural biology tools. Their recalcitrance to structural measurements is likely due to the very flexibility that allows them to perform their multiple functions, switching among functions and conformations rapidly to accommodate, evade and exploit the host organism.
IB: I was working with T. brucei already during my master studies, and I got the feeling that the research on the mitochondrial genome segregation machinery was at a turning point. Many (if not all) key players had been discovered, and yet many questions remained. Questions with answers I hope to find using structural biology approaches. Seeing something, often is an immense help in understanding how it works. And Structural biology allows us to see the structures of proteins and complexes we are investigating.
SK: I have always been fascinated by the intricate interactions between RNAs and proteins and their diverse cellular functions. I started my scientific career working on eukaryotic translation initiation and termination, then moved on to the ribosome assembly, and now study ribosome modifications and how they regulate ribosome’s function.
Is there an achievement or contribution in your career so far you are most proud of?
EA: I am most proud of being collaborative and helping my colleagues succeed either by helping them get started in crystallization and becoming crystallographers, or securing connections with institutes beyond ours where we can cross-train.
IB: Being an early career scientist – basically a baby in the field – I did not make any major discovery yet. But I am definitely proud of myself for how much I learned about structural biology in the past year.
SK: Throughout my career I have worked on challenging complexes, like eIF3 or pre-ribosomes, and I think my works have collectively helped their fields move forward. It is therefore hard for me to pick a particular one.
Why did you apply to the SPA course?
EA: I applied to the course to learn EM formally. I had been learning disconnected aspects of EM by making grids while others helped me screen them. I also shadowed colleagues to learn how to use our microscope or process the small datasets that we collected. I wanted a more rigorous, focused training session, and to learn from experts who could provide me and our lab with new insight into our current workflow.
IB: I applied for the course because I don’t have much experience in single particle cryoEM. I needed someone to show me how its done properly. Instead of having a colleague from my lab teach me everything, we decided it would be best if I learned in an organized setting. That way I could see the whole workflow within a short period of time, and I was made aware of details that might have gotten lost in a spontaneous teaching by a colleague. Especially aspects of reconstruction which can lead to wrong structures.
SK: Thanks to the recent technical advancements, cryoEM is now the super star of structural biology. Like many crystallographers these days, I am trying to learn cryoEM and found this course to provide a good combination of theory and practice on this subject.
What was the most useful thing you learned during the course?
EA: The course was the first time I processed data on my own, which happened during the cryoSPARC tutorial. I am now processing my own project data at home, and was able to jump in relatively easily. Before the workshop I was intimidated to begin processing because I just didn’t understand the workflow, or the rationale for choosing among many different types of workflow.
IB: The reconstruction on cryoSPARC was very helpful. Especially the fact that the reconstructions for most people didn’t work at first. It gave us the opportunity to troubleshoot, and see how some seemingly unimportant parameters may have big impacts on the end product.
SK: I learned many useful things from sample preparation to analysis and model building and refinement.
What was the biggest challenge you faced during the course?
EA: My biggest challenge also occurred during the cryoSPARC tutorial because it seemed as if my fellow students were more advanced than I was, so I stumbled at several steps along the tutorial whereas others could sail through what was more basic to them.
IB: Most of the course participants come from a crystallography background. Not having that background, was not a problem for most things we looked at in the course. I only noticed the difference when it came to modeling. It was much harder for me to follow, than it was for the people around me. They knew the programs at least a bit, while I had never used them before.
SK: Sometimes I felt the pace of the course is too fast, which is understandable as they were trying to fit as many things as possible in a short time.
What about the course surprised you the most?
EA: It was such a collegial environment where we were encouraged to get to know one another as colleagues. I had expected a less formal opportunity to work with others in our cohort, lecturers and tutors, and I’m glad for it! I suspect we’ve made some lifelong connections there.
IB: I was surprised how much I learned in just one week.
SK: What I was particularly fascinated by was the infrastructure at NYSBC.
Is there a particular moment or memory that stands out to you?
EA: The moment that Ed asked us to choose an image to be the backdrop of our class’s roundtable discussions. We chose an image of a virus because that week was the week when SARS-CoV-2 was first detected in New York, and when the ASBMB meeting as well as several other large conferences were cancelled weeks and months in advance due to the encroaching pandemic. Little did we know then that our gathering would be the last of its kind for many months.
IB: I remember the feeling when I saw a Titan Krios for the first time. In my home institution, the most powerful TEM we have is the Tecnai F20. I felt like a child looking at a huge pile of candy.
SK: Making ice cream with liquid nitrogen was fun. I also enjoyed the couple of time I got to spend time with the NCCAT staff after work.
What have you been doing since the course ended?
EA: The week I returned to California, our lab had one last group meeting before we began actively physically distancing from one another in response to the pandemic. Our new Krios and our recently relocated Titan Halo had been in the process of being built and reassembled, respectively, for several weeks already. Fortunately, just as our lab began assembling the foundation for our Coronavirus Immunotherapeutic Consortium (the CoVIC: https://www.lji.org/news-events/news/post/la-jolla-institute-for-immunology-to-host-coronavirus-immunotherapy-clearinghouse/ ) which will employ our microscopes and manage the worldwide search for the optimal immunotherapy for preventing and curing COVID-19. We are a lab working on the structures of viral proteins and antibodies against viruses that are among the most deadly in the world. So it was a great frustration and irony for us to halt our other work completely. Those of us who had been working on SARS-CoV-2 and the CoVIC projects continue to work actively in the lab, while the rest of us are mostly at home working on data analysis, including several cryoEM data sets.
IB: The corona crisis started in Switzerland while I was in New York. Case numbers increased rapidly, and the government implemented new restrictions almost daily. When I came back, I was already aware that at work I better not start longer experiments. I was in the lab for two weeks, before I was sent to work from home. Shops and restaurants are closed, and we are not allowed to meet in groups of more than 5 people. Luckily, all my friends and family are healthy.
SK: We have been at home for several weeks already. I have been busy analyzing the several terabytes of data I collected during the course.
Were there any skills you’ve learned that you’d like to apply to the work at your home institution?
EA: The workshop did an excellent job at reinforcing the importance of sample preparation. I made several grids within a week of returning to the lab from NYC. I included several new-to-me conditions (e.g. multi-blotting) I had learned at the workshop, for which I’m grateful. I am now processing a dataset that resulted from one of these grids.
IB: The processing part was completely new for me. With the practicals at the SPA course, I feel ready to play around and reconstruct things.
SK: I liked the tricks for model building we learned on the last day. I am also advocating for using cryoSPARC at my institute.
What would you tell someone who was thinking of applying for an NCCAT cross-training course?
EA: DO IT. Do it regardless of your level of expertise. It’s a treat to be tutored by so many experts in such a small teacher-to-student ratio. There’s always something new to learn in a field that’s growing so rapidly.
IB: Go for it, and take me with you!
SK: Definitely do it!!
Did you have a chance to explore New York City during your visit?
EA: I did after the meeting. I finally took in a comedy show, at the Comedy Cellar no less! I’d meant to do this since college in the early 2000s. Each time I return to NYC I also like to run around Central Park. I did manage several miles through and around it. I’m especially glad to have stopped at the Egyptian Obelisk in the park where I used to go often as a child when my family and I lived in Queens.
IB: Yes, I had plenty of time in NYC, as I arrived already on Wednesday of the week before the course. I am a big fan of the Bronx Zoo. Stalking squirrels at Central Park was really nice too. You can see the pattern here: I am happy wherever there are animals.
SK: Only on the afternoon before the course started I got to see Brooklyn bridge.
What do you enjoy doing when you’re not working?
EA: I have a new piano, so I’m able to return to regularly playing the piano. Running is my favorite form of exercise, so I explore on foot the trails and new neighborhoods around southern California as often as possible. I am also a mother of two children, ages 11 and 9 years, who currently live full-time in Philadelphia while I train in the Saphire lab in La Jolla. So I spend a considerable amount of time traveling back and forth between California and Pennsylvania.
IB: I’d say I have two main passions: Music and Gaming. In corona free years, I go to many metal concerts each year. And in summer, one can find me at several music festivals in Europe. I’m playing a lot of Dungeons and Dragons as well as some PC games.
SK: Playing with my son and reading.
Is there anything else you would like to share with me that readers might find interesting?
EA: Model building is my favorite part of structural biology. As a crystallographer I had many opportunities to build and validate models. In the years since I began my PhD (2012), cryoEM has evolved so rapidly that now I can apply my skills at model building even to cryoEM. This is a thing I didn’t realize then would be possible so soon!
SK: The staff at NCCAT are AMAZING!
Is there any question I should I have asked you, but did not?
EA: What technique or concept would you have liked to learn that you could not?
I would have loved a lesson on how to fill the ethane cup for plunge freezing with a Vitrobot! I never get it right the first time, and so have not yet done it independently.
*Some questions and answers have been edited for clarity and brevity