Scientist Stories: Eva Nogales, How Cryo-EM Informs Molecular Biology

Welcome to the Lessons from Lab and Life podcast. I'm your host, Lydia Morrison, and I hope our podcast offers you some new perspective. Today I'm joined by the brilliant Dr. Eva Nagales, a Howard Hughes Investigator and head of the Division of Biochemistry, Biophysics, and Structural Biology of the Department of Molecular and Cell Biology at the University of California, Berkeley., and most recently, Dr. Nogales was honored as the inaugural lecturer at the Donald G. Kome Honorary Lectureship here at New England Biolabs. Dr. Nogales has had an amazing career in the field of cryogenic electron microscopy, a technique that bridges the gap between molecules and cells, allowing the study of large protein complexes that operate within a living cell. Thank you so much for joining me today, Eva.

I'm truly delighted to be here with you.

I was hoping that you could tell our audience what cryo-EM is and how it informs biological studies.

Very good. So cryo-EM, which stands for cryo-electron microscopy, is a modality of this technique that has been used for many years to visualize objects at high resolution using electrons instead of light, but where the cryo refers to the fact that the sample is Frozen biological samples suffer from two weaknesses when it comes to being visualized by EM. One is that they are wet, they like to be in water. Water is incompatible with the vacuum that is inside electron microscopes, so we freeze them and we keep them at temperatures where ice can withstand those— that high vacuum. And the other is by freezing them, we protect them. If is a cryoprotectant against the radiation damage that is caused by the bombardment of biological samples with electrons. So it's a way to study biological samples and to do it at the highest possible resolution where light cannot take you.

And what was it about electron microscopy that drew you to this technical field?

So cryoelectron microscopy today stands as a new and very powerful alternative to structural biology techniques that have been used for many years to determine the atomic structure of proteins and nucleic acids. And those are nuclear magnetic resonance and especially, most especially, X-ray crystallography. So X-ray crystallography is a very powerful technique that, however, requires large amounts of samples to carry out crystallization trials, and you need your protein or your assembly to crystallize in order to then get diffraction data from which you can deduce the structure. Unfortunately, there are many samples that are completely resistant to crystallization, either because they cannot produce enough in enough amounts to do all those crystallization trials, or because people have tried and tried and they will not crystallize. It just so happened that the systems that I was interested in characterizing are of that type. One have to do with microtubules. They are a polymer that by definition is uncrystallizable, and the other are very large protein complexes involved in gene expression regulation that due to their size and due to their flexibility have resisted crystallization for many years. What attracted me to this technique was the fact that I could use this to visualize whatever it was that I was interested in. And to begin with, when this technique was not as powerful as it is today, it meant that I could be limited in resolution. But to me, it was more important to look at the full assembly as close as possible to how it is in the cell, even if I didn't have atomic details. Thankfully today, there's no such compromise. We can look at many samples and in most cases obtain the atomic resolution that allows us to understand them in great detail.

And how have electron microscopy studies helped define cytoskeletal interactions such as microtubules that you mentioned?

Yeah, so microtubules are these amazing polymers that are made of alpha-beta tubulin that associate together forming a tubular structure, therefore the name microtubule. Microtubules are present in all cells, all eukaryotic cells, so all the cells in your body, and they play many different roles. They are involved in organizing the contents of the cell. They are very important for chromosome segregation during cell division. Or they can be used to generate structures that actually propel cells like the flagella in a sperm. They do all of these functions because they have a property that is called dynamic instability by which they can switch between growing and shrinking phases. This process, although it can occur with pure tubulin, is highly regulated in the cell where microtubules interact with hundreds of different proteins. So looking at how these proteins interact with microtubules is a beautiful type of experiment, of goal, for which cryo-EM is perfectly suited. And we've been able to therefore visualize microtubules where tubulin is in assembled physiological state and see how different proteins bind to that microtubule surface, that lattice of different subunits, how some of them actually bind to the microtubule across a number of subunits serving, for example, as staples that keeps the microtubule together, or how some of them can recognize special features that are present at the end of microtubules. These are the studies that could not have been done with this resolution by any other methodology.

Are the microtubule samples that you are studying, are those come from endogenous sources or are those expressed in vitro?

Microtubules are pretty abundant in cells. They're particularly abundant in neurons. Mammalian brains, the brains of cows especially, pigs these days are fantastic sources. And there are great tricks to purify them from there. So a lot of the work that has been done with microtubules in vitro have been done from purified neuronal, you know, mammalian brains. From the native source. From the native source. And this was great, but on the other hand, it gave us very little playground to study mutations and to control what we had because tubulin exists in different isoforms. It has many different post-translational modifications. And when it's purified from cells, it comes at this, you know, very confusing mixture. And in fact, the difficult thing was to have an overexpression system because the folding of tubulin is incredibly complicated. And it was known, and it is an essential gene, you cannot overexpress or underexpress or express a mutant. For a long time, it was very difficult to have recombinant tubulin. It's only the last few years where a number of labs have pioneered the production of tubulin from insect cells, for example, and that studies of single isotypes or studies of mutants can be done. So we've done both. We've done many of our studies, which is this purified endogenous tubulin, but we've done more and more with specific isoforms from either human or yeast tubulin that have been purified, or, and including mutants that we chose, you know, in a specific manner to prove specific functions of tubulin. Both, but the default for many years have been endogenous sources.

Do you feel like both offer really interesting insights into the function of microtubules, or do you feel like the ability to overexpress in the last few years has really allowed you to hone in on very specific questions and problems?

I think that it's great to have this source of cheap material, but we are opening new doors to new questions now that we have access to recombinant tubulins where we have a lot more control and we can be more incisive in the questions that we're able to pose and answer with this new material.

That's very interesting. How has your lab used structural studies to elucidate gene expression and and regulation?

Yeah, so we study the molecular machinery that is involved in regulating the expression of proteins. This is a process that starts with transcription of protein coding genes by RNA polymerase. RNA polymerase II in particular, it is a very large complex of 12 proteins. It's half a megadalton in size. And it's wonderful that it can, copy DNA into mRNA, but is otherwise a very stupid enzyme that doesn't know how to find the beginning of a gene or how to open the duplex DNA to get access to the transcribed strand. To do this, it requires a cohort of other factors that have to come together into what is called a transcription pre-initiation complex around the start site of a gene that is marked by specific sequences, that are recognized by some of these factors. That pre-initiation complex also includes factors that have ATPase activity that is used to generate work that is used for the opening of the duplex DNA. These are all very large complexes that come together forming an assembly of about 3 megadaltons in size. They are not in Some cases, there isn't any expression system. We do have to rely on endogenous materials. Most work has been done either with human in our case, or otherwise with yeast, budding yeast proteins. And really cryo-EM has been uniquely suited to study it because we get them in very small amounts. They really resist crystallization. And in many cases are dramatically flexible. Maybe that's why visualization would be so difficult. Cryo-electron microscopy has been able now to describe the structure of all these components separately and coming together, coming together onto the DNA right by the transcription start site. We've been able to get an amazing amount of biological insight by directly visualizing different stages in the process of the transcription initiation process.

And of course, because this is such a large macromolecule, these are the kind of, um, this is the kind of resolution that you wouldn't be able to ever see with an X-ray crystallography study, right?

When samples can be crystallized, depending on the quality of the crystals, it may be possible to obtain structures that are at resolution as fantastic as 1 angstrom, or that the crystals are limited and the resolution, the order of the crystals or the size of the crystals is limited and the resolution doesn't go past 5 or 6 angstrom. It is a little bit the same with cryo-electron microscopy, depending in the case in the behavior of the sample, how stable they are, in how many different conformations they exist when we are looking at them. That it is possible to get below 2 angstrom resolution or get a stack at 9 or 10 angstrom resolution. So for these large macromolecular assemblies, there are regions that are very stable, typically in the core of the structure, and those we can see with basically atomic definition. While there are regions on the outside that tends to be the more mobile, the arms, the legs of the structure, where the resolution can be more limited. But in all cases, we're able to describe the range of motions and that information, the dynamics of this molecule is a new dimension of information that you cannot get when you have a single static picture. So there's typically any structure of something that is as large as 1 megadalton have different resolution regimes. Some parts of the structure are very well defined and other are a little fuzzier and we see less well, but we can tell that it moves and hopefully we can describe the range of motion, which is just as important as the details. At atomic level.

And how do you deal with the challenge of those flexible regions?

So when the sample exists in a heterogeneous state, either because there's different compositions, for example, there could be a factor that comes and goes from the main complex that engages and disengages, so there are different states compositionally, or especially when you have conformational heterogeneity, meaning there are regions of motion within the complex, is going to involve— is going to require a lot more data and very complicated computational approaches to be able to separate the states, zoom in one area, and push the resolution maybe one area at a time. So more flexibility, more complexity, means more data and more computation.

And what are the implications of this knowledge, of the knowledge about the factors included in the initiation complex and the binding order and those flexible regions?

So, you know, I'm a structural biologist and I find it hard to believe that people will need convincing of how important it is for us to know the physics and the chemistry of biological molecules, what they're doing, their function. So we want to understand how biology works, how we are alive, how we are what we are, starting from relatively simple molecules. They're not, they're actually complicated molecules, but that build up ultimately the complexity of the cell, of tissues, and organisms. And the details of how they interact with one another, how they collide, how they move, how they push, how they rearrange. It is critical for us to have an understanding of these processes, which to me is like knowing the blueprints of how we are made in the, say, correct way, so that when things go wrong, when things fail, when there are mutations, whether diseases, we can understand what they mean at the molecular level and how we can target them. How can we design therapies, small molecules, gene therapies that will correct from those defects? But how can you fix something if you don't know how it works, how it mates, how all the pieces come together? So that kind of knowledge is just critical, not just as a fundamental knowledge, just like we want to know the particles of the universe and how galaxies are made, although they don't necessarily have a practical, you know, purpose for our lives. So that exists, that wanting to, you know, being curious about how we are made and what biological life grows out of is important. In the case of biological studies, there's always a biomedical component. It's always the fact that this is a requirement and a basic starting step towards understanding disease and fighting it. So you can imagine many diseases have to do with, you know, overexpression or underexpression of certain cellular components that are required for, you know, the, the well-being of the cell or to be able to react to an infection, things like that. And if you don't understand how genes are expressed in the first place, how can you modify or understand how that gets dysregulated and how it is possible to fix it? So I think these motions, these interactions, they are part critical parts of the story of how we are made, how our genes are read, which are, you know, absolutely basic for understanding cell biological processes. So I think we are, we're working on something important.

I think so too. Can you tell us about the story of your work on TF2D?

So, so TF2D stands for transcription factor that works with polymerase 2, and the D, I understand, for the person that discovered it, was just the order things were being discovered and being called A, B, C, D. In any case, this factor is actually a complex of about 14 different proteins. It has a huge size at the molecular level, 1.3 megadaltons. And it has a number of very important roles in gene expression, in the reading of DNA into messenger RNA, in the reading of a gene. And the first function is that it has to be able to recognize regions in the genome that correspond to the beginning of a gene, the start of the gene where the polymerase needs to start reading the message.. And then once it has recognized, bound to those regions, it has to have the cap. It recruits the rest of the factors, including the polymerase, that are required for beginning the copy, uh, the copying of the DNA into messenger RNA. And it regulates how much mRNA is being produced through the— in its interaction with gene-specific proteins, transcription factors that act at the end of a signaling cascade to respond to the need of the cell that tells this particular gene has to be produced now in large amount, or it has to be otherwise shut down because it's no longer needed, or, or this was the wrong thing to be producing. And TF2D is able to interact with these gene-specific transcription factors that upregulate or downregulate transcription, and in doing so, probably change both its capacity to bind to DNA and its capacity to recruit the polymerase to therefore regulate gene expression. It does this very complicated set of functions by having a large architecture that includes mobile regions that are able to respond to different signals to provide different outputs in terms of affinity binding to the DNA and capacity to recruit the polymerase.

So, now that you've done such incredible work to help elucidate sort of the complexities of that pre-initiation complex, what's next for your work on gene expression? Right.

So, you know, I think something that is fascinating about biology and that we as humans have yet to be able to imitate to the same degree is the capacity to adapt and the capacity to add regulatory layers. I think many of those hearing this podcast will know that the number of genes that a human being has compared to I don't know, a plant or a budding yeast unicellular organism is not very large, but they're able to use that genetic material in a way that gives them a lot of flexibility and that allows for further evolution of more and more regulatory processes. So this at the molecular level occurs just like it occurs at the organismal level, So far, the studies that we have done on TF2D are of the most basic aspects that have to do with the DNA recognition and a little bit on what it involves to recruit the polymerase to the site. But how its activity is regulated in a larger context where there are other factors that are involved, where it's not acting just on DNA but on chromatin, which is a much more complicated a structure where DNA is wrapped around nucleosomes that have different colors, if you want, that has to do with the way the histone proteins that make the nucleosome and wrap the DNA have been modified and painted in different ways. They all act to regulate gene expression. TF2D is interacting with all these different factors and those, that level of added complexity of regulatory elements, that is at the heart of the regulation that allows cells to adapt and cells to become, you know, respond to the environment or the needs of the organism. That's what we are starting to study now. We started with the basic and now how is all of these regulated and controlled? How is this activity increased or decreased? This is what we will be we will be doing now and we will be doing it in the context of chromatin that most closely resembles what it has to face, what is in the nucleus of each one of our cells.

That's very interesting. I look forward to seeing more work from your group about that because it is amazing to think about how the size of the number of genes encoded in our DNA is not that different from far more simplistic organisms, and that those subtle molecular changes are what's really regulating the differences between humans and plants. How important are collaborations to the success of your research?

Right. You know, sometimes I get really amused by the way Hollywood portrayed Scientists, I think all scientists do. There's one, you know, it makes me feel that, you know, the way they portray other professions is probably just as flawed. But, you know, scientists are either these malicious, crazy scientists that are set there to destroy or dominate the world, which honestly rarely happens. And the other is that, you know, you have this single a beautiful girl or single handsome man that one day, just having coffee, starts scribbling something in a napkin, in a paper napkin, and come out with a solution to the energy crisis in the world. Nothing happens like that. Science is by definition collaborative. It is a team effort. It's a team effort from the point of a single project having several people working at the— from different angles with different expertise, at the level of laboratories where different projects are intercalated and people pass along their experience and their know-how and their opinion and their advice and criticism. And then it goes beyond labs, and it is very common that scientists interact with one another, and they do it across universities, they do it across countries and across continents. And it's the beauty of science. And, you know, everybody has to realize that, you know, to become a scientist you have to study for many, many years. You become typically quite specialized because we are, you know, getting more and more detailed, and it's harder and harder to, to push the barriers of our knowledge. And it is really fantastic to be able to to interact with other people that have been focusing on different things or in, or trying to answer similar questions with different tools, and how that coming together has a value that goes beyond the sum of the parts, that is synergistic, I think is a word that we love to use. So collaborations have been very important in my life and my scientific career, In some cases through a collaboration I've opened completely new areas of research in my lab that then has become, you know, mainstream, but at a certain point it was just someone approaching me and telling me, you know, look at this that I'm studying, it's so interesting, could we work together on it? And then they become really part of my scientific life. To situations in which I have questions that my own expertise I cannot answer and I can team up with someone. It's just, uh, it is one of the, my most favorite, um, aspects of, of my job. The fact that I feel unlimited to whom I can approach, uh, and who I, whom I can engage, uh, to get science done. And we were talking through the day about many different things, and one of the things that we mentioned is that I think scientists will make for wonderful diplomats. They judge each other by their intelligence, thoughtfulness, dedication, and ultimately their results and the strength and the rigor of their science, not by any other means. They are able to take criticism, they learn from failure, and, you know, they really love to work without barriers. We collaborate with other places. We bring into our labs people from all countries and all cultures, and the richness of that mixture cannot be understated.

I think you're absolutely right, and I think that global scientific collaborations are really what has driven some truly powerful scientific breakthroughs. It's wonderful to see that. I think you're absolutely right that the scientists would tend to be good diplomats as well. You've mentored so many individuals at various stages in their career throughout your own career. I'm curious what advice you would have for girls and women who are pursuing careers in science.

Mhm. Yeah, so I will tell them that I would not be happy— I can't imagine being happier doing anything else. That I, I hopefully has come through as I answer these, your previous questions. Uh, I feel very passionate about what I do. Every day is different, and my day involves interacting with young, intelligent people and learning every day. So what can be better? But it is true that science can be frustrating because you are pushing the limit of knowledge with the limit of the technology that is available to you at any given time. And we do have to deal with failure all the time. So it is very important that you come to science with self-esteem, with appreciation of who you are and your capabilities, that you don't take failure as personal failure, but as an opportunity to learn and to realize that you are really moving that barrier of knowledge and breaking new ground. So my recommendation will be, you know, Let your curiosity and your wish to make discovery drive you and energize you. Be resilient in the face of failure because it's going to happen. We have to deal with it all the time and repeat experiments and rethink our questions. You know, destroy our models and start over again. Um, and just believe in yourself and don't be ashamed of— show off your, your capacity. Um, and don't fall down into the self-doubt well for more than is required to just spark new ideas and while being humble, you know, being in pursuit of what you're passionate about. So be resilient, be enthusiastic and passionate, and don't let anybody tell you that you cannot do it.

Then you'll do fine. Dr. Ivonne Nogales, it has been a pleasure and a joy to have you here today. Thank you so much for joining us.

Thank you so much for having me.

Thanks for joining us for this episode of our podcast. As always, check out the transcript for links to further resources. And please tune in next episode when the mentors and team members from the Genes in Space program are here to share how aspiring high school students are awarded the opportunity to send their experiments to the International Space Station.