We recently connected with Dr. Paolo Swuec, the facility manager at the cryo-EM lab at the University of Milan in Italy. We wanted to learn more about his work using single particle, high-resolution cryo-electron microscopy to study the structure and function of diseases. Read through our interview to discover what he’s currently working on, the everyday challenges he faces with cryo-EM, and how his hobbies support his work at the microscope. Watch the full interview to hear our discussion, or keep scrolling to read a snippet of our conversation.
This Q&A has been edited for clarity and length.
Can you tell us more about you and what you do?
My name is Paolo Swuec, and I’m the facility manager of the Cryo-Electron Microscope Lab at the University of Milan. We are interested in investigating the structure and the function of macromolecular complexes such as viruses, membrane proteins, and other enzymes in order to get a better understanding of their activity, and also, more importantly, to understand what goes wrong in a disease scenario.
By training, I am a pharmaceutical chemist who got involved with a bit of computational chemistry and finally made it to protein biochemistry. I got into the production and the characterization of macromolecular complexes, and in 2011, started adopting single particle electron microscopy.
Why did you choose cryo-EM single particle analysis?
I opt for single particle high-resolution cryo-electron microscopy mainly because we can use low quantity of samples and get a fast response. That means that, when we get the purified sample, we always get an answer from the microscope. Either the sample needs to be optimized, or it’s ready for a high-resolution characterization.
Another reason why we opt for single particle cryo-electron microscopy is because of its versatility. We can investigate a wide range of different complexes, from big viruses to small gene-editing complexes. We tend to work a lot with membrane proteins as well as amyloid filaments. For example, our latest research paper was on the architectural study of cardiac amyloid fibrils that were directly extracted from the tissue of a patient.
What inspired you to begin working on the biology of amyloid cardiomyopathy in general?
Amyloidosis is the result of a misfolding process. In particular, we are studying cardiac light chain amyloid fibrils that undergo a misfolding process from the light chain into cardiac tissue. The issue is that the light chain of antibodies get mutated and go through a misfolding process that will start building up thousands or millions of copies of this mutated light chain in a different combination. And this will build up as a filament. Now, the filament could impair the natural processes of the cell and eventually leads to the disease (cardiac amyloidosis).
When I first came across this project, it was a bit of an issue because everything was based on recombinant models. So you could not really get an understanding of the sample directly from a pathological situation. Thanks to cryo-EM and single particle analysis, we can directly extract those filaments from the heart of a patient who died from amyloidosis and get high-resolution information about the structure and the overall architecture of the filament.
What challenges do you face in your research?
The current challenges that we face every day are more related to specimen preparation. In order to maximize the chances of a successful single particle experiment, you really need to start by optimizing your sample. To do this, we need to get vitrified ice as thin as possible in order to maximize the signal that will increase the chances of getting a high resolution structure.
The second challenge is related to how dynamic your enzyme or complex of interest is. It is very difficult for us to image something that is moving very fast. In the best scenario, where we have a complex that has distinct conformational states, we can get a high-resolution structure from each of the different confirmation from the same data set. That means that we can save time and money while doubling our results.
How has cryo-electron microscopy benefited your research?
Cryo-EM helped us directly investigate the structures of these filaments by looking at not a model or a recombinantly expressed system but directly at the sample extracted from the heart of a patient. What is special about this kind of characterization? It’s not just the resolution but the fact that we are directly looking at what went wrong in the diseased tissue.
Now that we know the structure of the amyloid fibril, we can go back and get a better understanding of the whole process, so how we got to the final filament from, let’s say, the parental light chain. Currently, we are mainly focused on single particle analysis, and while we are slowly opening to in situ tomography, I hope to soon apply micro electron diffraction (MicroED) on one of our microscopes.
What excites you when working with cryo-electron microscopes?
The feeling of having a direct look at a protein in the microscope is unbelievable. To think that you can have a direct look at your sample is incredibly satisfying. But you know what’s fun? Astrophysicists investigate the incredibly big stuff, while we investigate not incredibly small, but small stuff. So when you do compare the colliding galaxies to cell division, the images look the same. It’s fantastic. It’s like the symmetry of life, and we are in the middle. There’s something there. It’s fantastic.
What makes your lab stand out from the others?
So far, we are the only high resolution cryo-EM lab in Italy. That means that we have a huge responsibility for the scientific community. And we have an open-door policy, so we tend to share all the protocols and methods. For this reason, we had two practical workshops within the same year. We tried to select participants from all over Italy in order to share in our expertise and knowledge of the field, in order to build up a better cryo-EM community. We have many collaborations in Italy and outside Europe, including with Columbia University as well as internal research groups here at the University of Milan.
Outside of cryo-EM, what are your hobbies?
When I’m not at the microscope, I am actually taking pictures not of viruses and enzymes but of everyday life. I do that to start up my creativity and because it’s useful in my experiments. So do all the other hobbies that I have, such as music. It’s another thing that can use to foster my creativity.
Steve Reyntjens is director, product marketing at Thermo Fisher Scientific.
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