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intro/outro (00:01):
Welcome to Cryo-Talk a Bitesizebio Podcast, sponsored by Thermo Fisher Scientific featuring conversations between your host, Eva Amsen and experts in the field of Cryo-electron Microscopy.

Eva Amsen (00:15):
Today on Cryo-Talk we are joined by Eva Nogales distinguished Professor of Biochemistry, Biophysics, and Structural Biology at UC Berkeley, as well as a Senior Faculty Scientist at Lawrence Berkeley National Laboratory and Howard Hughes Medical Institute Investigator. We're discussing some surprising discoveries in her research career.

Eva Nogales (00:35):
And when this happened, it really took a turn to get us in the direction of finding something that was truly biologically meaningful.

Eva Amsen (00:43):
Why it's important to work with people you like.

Eva Nogales (00:46):
Going through those ups and downs. It is very important to be surrounded by people that you feel you can trust.

Eva Amsen (00:53):
A sabbatical in Spain.

Eva Nogales (00:54):
And I am at the, uh, CNIO, the National Cancer Center in Madrid. So very close to my hometown.

Eva Amsen (01:01):
And her love of music.

Eva Nogales (01:03):
I actually have tickets for a, for a rock concert here in Madrid. One of these massive things I've never been to really one.

Eva Amsen (01:11):
All in this episode of Cryo-Talk.

Eva Amsen (01:24):
Hi, I'm Eva Amsen. Welcome to Cryo-Talk. I'm here today with Eva Nogales. She is a distinguished Professor of Biochemistry, Biophysics, and Structural Biology at UC Berkeley, as well as a Senior Faculty Scientist at the Molecular Biophysics and Integrative Bioimaging Division of Lawrence Berkeley National Laboratory, and a Howard Hughes Medical Institute Investigator. So Eva, how are you today?

Eva Nogales (01:51):
I'm great. How, how are you doing?

Eva Amsen (01:53):
I'm good too. Thanks for joining me here. So before we get started, um, can you tell me a little bit about your research background? So what did you do before you got to where you are today?

Eva Nogales (02:06):
Yeah, sure. So I was an Undergraduate Physics student in, uh, Madrid where I'm from at the Universidad Autonoma de Madrid. And then, um, I was very fortunate to be able to do my PhD at the British Synchrotron at the Synchrotron Radiation Source at Daresbury Lab with Joanne Borders. And that was an opportunity for me to combine a small angle x-ray solutions scattering with Cryo Electron Microscopy, to study, Tubulin Assembly, and effect of Antimitotic Drugs. And, um, that in terms gave me the right background to apply for a Postdoc that in and turned out to be the most fortunate things in my, in my career, uh, at, uh, the Lawrence Berkeley National Lab with Ken Downing and with, uh, with him and Sharon Wolf, we worked using Electron Crystallogrophy to obtain the first structure of tubuling, um, which, um, is something that I've been working on since my PhD and I still work on. And that worked out very well for all of us and, um, is what landed me the position at UC Berkeley, where I've been ever since.

Eva Amsen (03:18):
Great. And, and how did you get, um, you mentioned you used, EM, how did you get from EM to Cryo-EM and how have you used that in your work?

Eva Nogales (03:26):
Right, right. So, um, so I was, um, I, when I refer to EM, I'm, I'm talking about Cryo Electron Microscopy, Negative Stain, a combination of both, uh, but mostly Cryo Electron Microscopy ultimately. So my first use of Cryo-EM was very, um, unusual because as I briefly mentioned, I was doing a small angle x-ray scattering and this was timed result. So the idea was to have tubuling that was in the process of assembling or changing confirmation, typically with temperature, something that we could vary and then see the progression in time. And, uh, x-ray scattering is a technique where it is not trivial to, uh, to analyze cause it's, it's a single curve, uh, decay curve and the meaning of, of it and the changes in some of the features, um, we could, uh, interpret a little bit better by obtaining, um, images of maybe one of the states that we could then fully transform rotationally average and compare with SAXS data, believe it or not. And that allowed me to interpret what certain peaks in the scattering meant. And when they change and knew what parameter in the features of the object were changing, but ultimately it told me, you know, maybe I should concentrate on Cryo-EM cause it's a lot more informative and not throwing away information, but rather the opposite. So, uh, after that, what I was using somehow a quantitative use of the images, but not for three dimensional reconstruction, which is what we now understand as Cryo-EM. Uh, when I moved to, um, Ken Downing Lab, what we did was Electron Crystallography, which is a special modality, um, of Electron Microscopy, uh, and that had really been developed at the LMB and in particular Richard Henderson had obtained in 1990, the structure of bacterial adaption using such methodology and is one in which images of two dimensional crystals are combined with Electron Diffraction. Uh, so for the efficient others, we will use the diffractions to calculate the amplitudes of the structure factors and then combine with the faces of the images and then combine many images to get back, um, to the full free space, fully transfer back and get this structure long story short Electron, uh, Crystallography used to be in the Nineties, um, kind of the, the technique, the ruling technique, if you wanted to obtain atomic structures with electrons using electron microscopes, but obviously eventually, um, the Single Particle Methodology that people like, uh, Joachim Frank, um, were able to develop, uh, took over and the, and the rest is history. Now, Eelectron Crystalography is hardly used, uh, at all. There's a, there's a different thing that has to do with Microdiffraction, uh, which is probably not what I want to be talking about right now. This is not something that I use, but, uh, but that was my path, you know, first a very esoteric use of the images for my particular means then Electron Crystallography and then single particle Cryo-EM, the mainstream Cryo-EM where you don't need to crystallize your sample of interest to obtain a three-diional reconstruction.

Eva Amsen (06:54):
Yeah. And, and that's, um, not having to crystallize, does that make it easy to look at things like microtubule.

Eva Nogales (07:01):
Right. So, so microtubules are another example of the perfect sample for Cryo Electron Microscopy, um, because they're polymers they're large, they're repetitive. So Cryo-EM is, is ideally suited for it. While on the other hand, they are a real impediment for crystallization, in fact, um, tubulin, which is the protein that makes up microtubules, uh, has been also after we obtain the Electron Crystallography structure, which was of an, um, you know, an a, a rare, a strange self-assembly form of tubulin that forms in the presence of zinc ions um, and kind of aberrant assembly. Um, that was the kind of two dimensional crystal that we were able to obtain. After that two years later, there was an x-ray a structure of tubulin in a depolymerized state where the assembly of tubulin has been inhibited, but, uh, because microtubules cannot, would not be crystallized, polymers cannot be crystallized. Um, but they are indeed ideal and many labs, uh, through the years have used Cryo Electron Microscopy from the pioneer days of Ron Milligan, Linda Amos, Dick Wade, uh, for the study of microtubules, which my lab has continued to do push to high resolution, has studied many different combination states bound to antimitotic drugs, uh, bound to different, um, microtubule associated factors. So ideal Cryo-EM sample just like acting, um, and an actomy in filaments and things like that.

Eva Amsen (08:44):
Yeah. That's really a perfect technique for looking at those long structures. um, and what are you most excited about, um, seeing in the Cryo-EM field in the next like five to 10 years or so?

Eva Nogales (08:58):
Right. I think the most exciting thing is to build up complexity. So, um, this can be done in a number of ways. So right now, when we think of Cryo-EM, we are thinking of single particle Cryo-EM, and high resolution. And that starts typically with highly purified samples, just like as if we were gonna do crystallography, but without having to crystallize it. So with, with less in terms of requirements of, of amount, for example, which is already a, a huge deal. Um, but, um, the, I think something that will be very exciting is to make those reconstituted systems more complex, where we look at the interaction of many different molecular partners, uh, that will exist at a certain equilibrium, uh, both in terms of composition and, and confirmation. This is something that we are already starting to do. And I think the complexity can increase more and more, but, uh, obviously, uh, the, the real complexity complexity comes from looking at things inside the cell in, in, in the real, um, biological media, where they are supposed to act. And what I also very excited about is anything that happens in between, because now there's a lot of people that are putting a lot of effort into looking at extracts, uh, of different types of Cell Fractionation. Um, these are samples that have been very wonderfully used over the years for biochemical, uh, characterization of complex cellular pathways that I think we could also use. There are an intermediate between the single particle and the true cellular media that requires, um, techniques for thinning and where it is harder to manipulate the system. Where in, um, in something like an extract, you could deplete, you could add exogenous protein in excess or a certain label and things like that. And it still make mimic, make certain aspects. So building complexity, um, so that we get at the regulatory mechanisms and through pathways. So we go from individual molecules to many that interact in a dynamic way. And in some cases, even inside the cell where the context, the cellular context of the position of the different organelles, um, is, is, is very relevant, is where all the excitement, I think it is for me. So a lot more complex systems to answer a lot more complex biological questions.

Eva Amsen (11:32):
Yeah, yeah. It, we are really getting much closer to actually like seeing what really happens like in the cell between cells and not just a derivation it's like really looking at it. It's, it's very exciting. Um, now you told us a little bit earlier about your, your research and your career, and has there ever been a moment where your research or career took a surprising turn?

Eva Nogales (11:56):
Mm, well, I, I, I remember one particular moment where something that had really puzzled us for a long time, suddenly made sense and sent us on a path that ultimately, uh, have meant a lot of, uh, mechanistic insight that we, we were not, um, you know, expected to, to, to be able to, to obtain and, and that it will go that way. Um, maybe there will be others, but this is the one that comes to mind first. And it had to do with the study of a very large complex, uh, that we started studying, um, right after, you know, the obtaining the structure of tubulin during my Postdoc, I was, um, working on other tubulin related, uh, projects, but in the context of now my faculty position at UC Berkeley, I started collaborating with Robert Tjian, who is a world expert in human, um, gene transcription. And, uh, he got me interested in a complex that is called TFIID this is a very large 1.3 megadalton complex, uh, extremely difficult to reconstitute. We had relied on endogenous material and is present in very, very small amounts in the cell. And when we started studying it, we, you know, this was in the old days where Cryo was still kind of in its infancy concerning single particle methodology. And we knew that it, it was made of three main lobes. So this huge thing, which is describing a three lobe, um, complex, right? So this gives you an idea of the very low resolution that we had at the time. Uh, but we knew that this complex bound to two, sorry, bound to DNA, this is one major thing that it does. Um, and when we tried to visualize it bound to DNA, we could see into dimensional image analysis that it was there. And there was a line that really looked like the DNA. But when we tried to do the reconstruction and study from what we knew already of the complex, without DNA in, we, we never convert to anything that had DNA. And then eventually through very careful analysis of two dimensional class averages, my student, uh, Mike Cianfrocco at the time. Now he has his own lab at University of Michigan realised that one of the lobes was moving very dramatically, across the complex. So this was something huge, almost half a megadalton in size, and he was moving 150 Armstrong. And that was amazing because we, we just, we didn't quite realize the three lobes were about the same. And we, we just, uh, didn't know at, at that time. And the most important thing is that when he, uh, added DNA, the population of where the, the, that particular lobe went shifted, um, towards binding of the DNA, and this was kind of a revelation on one hand, the complex had had given us such a hard time, but being so flexible, I still know very few things that are that flexible, that people have described so far, but it had to do with the binding of DNA and eventually, you know, with the new detectors and the new methodology, we were able to push the resolution of the complex of 10 [inaudible] in different functional states. And he told us a lot, but we really didn't give up. It could we were that very close because it thought it was an impossibly difficult project. And when this happened, it really took a turn to get us in the direction of finding something that was truly biologically meaningful.

Eva Amsen (15:40):
Yeah. Wow. So, so that's a great story.

Eva Nogales (15:42):
Nature is difficult, but, uh, once you get to understand it, it had a reason for it.

Eva Amsen (15:48):
Yeah. , that's a good, good lesson to learn. . Um, now I was, we, we already talked a bit before recording and I was going to ask you if you ever went back to Spain, but, um, maybe you should tell people where you are now.

Eva Nogales (16:03):
Yeah, yeah. So right now I'm in Spain. Um, I'm, I'm taking a semester of sabbatical and I have been, uh, first, uh, in Barcelona at the Center for Genomic Regulation, just by the beach. It was really, really nice. And that was, um, actually, um, something that was facilitated that by the Valley Foundation who actually gave me a visiting professorship. So that was really, really nice of them. Now I'm also being subsidized, if you want, by another foundation, the Jesus Serra and I am at the CNIO the National Cancer Center in Madrid. So very close to my hometown from where I commute, um, every morning. So having a great time talking to people here in the CNIO that also do Cryo Electron Microscopy, like Oscar Llorca and, uh, and Rafael Leiro. Um, and it was also great, uh, in Barcelona where I had a chance to start a number of, uh, collaborations with, uh, with people like Thomas [inaudible] or like [inaudible] in microtubules and Polycomb complexes. So very, truly fantastic, um, sabbatical that is been, uh, you know, very, you know, a, a, a great time to think, and to start new things right now, and concentrating on our review, uh, that was commissioned by a journal, um, to Julia Mahamid and myself, uh, where we are trying to, um, see in which way single particle Cryo Electron Microscopy and Cryo Electron Tomography of cells, uh, intersect and, and, um, what the future may depart or, uh, may bring us and having a great time, you know, talking to Julia, uh, about this hopefully will get it finished on time.

Eva Amsen (17:58):
Yeah. I look forward to reading that when it comes out and I guess you must also just be having a good time being back in Spain, if you still have friends and family there. .

Eva Nogales (18:07):
Yeah. So I'm actually, I'm actually staying with my mother who's, uh, 86 year old. So, uh, really giving my brother a little break, uh, looking after looking after her and otherwise just, um, spending time with, uh, with my brother and, and his family and my husband and kids are gonna be coming in August. And at that point, my sabbatical will be over, but it will be a time to, uh, to do some traveling, both in England. My husband is from the UK. Uh, actually we are going to go to Wales, um, and, uh, and here in Spain, where we'll go to the north escaping the heat.

Eva Amsen (18:46):
Yeah. yeah. August will be a bit hot in the south um, so I've just got a, a round of quick fire questions for you. Yeah. So just give me a rapid answer. Um, do you prefer the city or the countryside?

Eva Nogales (19:03):
I love the countryside that is not so far away that if I want to go to a museum or a concert, I can, uh, take some public transportation on or, or a car, but otherwise the rest of the time I'm in a place that is peaceful.

Eva Amsen (19:17):

Eva Nogales (19:19):
I just, just realize that I'm in Madrid right now. And tomorrow the NATO conference starts, they have told us to stay home because apart from anything else, Biden is gonna come with 50 vehicles and is gonna block the main country in Madrid going up and down. So being back in my hometown, which is 20 miles north of Madrid, um, maybe telecommuting tomorrow. Yeah. That would be nice.

Eva Amsen (19:46):
it's a good place to be then. Um, do you like to cook?

Eva Nogales (19:51):
Yeah. I love to cook and I can do Spanish food. I can do some Indian food, some Chinese, although I like eating, eating when other people cook even more, if they're good cooks.

Eva Amsen (20:04):
What's your favorite thing to make?

Eva Nogales (20:06):
Um, you know, I love making paella and Spanish omelet cause I do it very well, but I, I, there's a chicken vegetable curry that I make for my husband. That is also very good. Um, but my favorite thing is do a big roast to roast the chicken and lots of vegetables and things like that. A lot of roast.

Eva Amsen (20:26):
That sounds great. yeah. Um, do you like reading and do you have any books to recommend?

Eva Nogales (20:32):
I, I love reading and since the pandemic I've been reading more than more than I wouldn't say more than ever. Cause I, I really read a lot before I became a PI and, and uh, and um, a life and a mother life just didn't, uh, give me much time, but during the pandemic I've been reading a lot and here during my sabbatical team reading a lot, I've been reading a lot of Spanish literature I have to, and, and, and also, uh, um, essays and things like that. So I do not know that everybody's gonna be able to get it, but, um, there is a particular book in Spanish it's called El infinito en un junco. So junco is like a, a particular type of, of, um, growth, you know, like a, like a bush of some sort from which, um, Papyrus the, the special, uh, pulp was used for making Papyrus in, in old Egypt. And it's about the story of the, of books, how they came to be. So is, is the, is the history of writing. And now how that writing, um, you know, was transmitted and how it changed in form and is, is, is written in a beautiful way that really relates to things that are happening to us today, like the internet and how communication and information transmitted. And then she relates it constantly to different other books, other essays, literature, movies. So I got to see the movie, um, Wings of Desire, for example, uh, I think in German and Spanish is more translated like the sky over Berlin, but in any case, just, um, and just is something that you really, you like it, but also it send you in different ways. That is a very, yeah. Uh, interesting.

Eva Amsen (22:27):
No, that sounds interesting. Um, yeah, my next question is actually about films. Is there a film or a TV show that you've enjoyed this past year?

Eva Nogales (22:37):
You know, I just, I just want to, uh, I just watched a movie that I really loved. It came out in 2019. It's very unique. I thought it's called Roma. Uh, it's actually, uh, Mexican, it's a movie about Mexico City in 1971, and it tells the story of a middle upper class in, in Mexico City, but all, uh, center around the, the, the servant, the people that are actually looking after the, you know, cleaning the house and looking after the kids. And, uh, and it, it just seems to be made of memories maybe of the director. Um, and it, it has a lot of, uh, events. It, it, you know, it, it tells the story of these, of these people and the, the, the tragedies of life that they're going through. But also in the context of what, what is happening there, there was a earthquake, there was a certain point, there was a fire.

(23:33):
There was a, um, you know, a student, uh, University student's demonstration that ended up, um, with people being killed, you know, in this demonstration, it's just beautiful. And it's done with these long takes where a lot is happening and in today's filmography, you know, there will be lots of changes of cameras and things. And this is not, this is like recording life very, very long, uh, takes where the camera doesn't move, but the true drama is there and you don't need the camera to be telling you how to feel or anything like that. The camera is just there to, to give it to you. Anything black and white, that really takes you back to the 19 early 1970s. Yeah. It is really, really beautiful. Beautiful.

Eva Amsen (24:22):
That sounds interesting. Roma, gotta check that out then. um, and do you like music?

Eva Nogales (24:28):
Yeah. Yeah. I love music. I love, uh, classical music. I also love pop music, um, and, uh, and having a mixture. So right now I'm listening a lot to DireStraights. Um, for, for example, um, a little kind of melancholic. That's also part of being in Spain makes me want to listen to things that I was listening when I was in, you know, a teenager or in my early twenties. . Um, but yeah, I love music. I actually have tickets for a, for a rock concert here in Madrid. One of these massive things. I've never been to really one or maybe one Rolling Stones in the US many, many years ago. So I'm very excited, a little scared. Um, but, but that'd be fun. A lot of fun. Yeah. Um, yeah.

Eva Amsen (25:17):
Um, and my last quick fire question is if you were not a scientist, what would you be?

Eva Nogales (25:24):
Yeah. You know,

Eva Amsen (25:27):
An alternate reality.

Eva Nogales (25:30):
I find it incredibly hard to think of what I would be because I really, I love being a scientist. I, I had even a hard time when I think of my kids, the fact that they're not gonna be scientists, well, maybe one ends up being an environmental scientist. The other one is, is, is doing engineering. And I, it is hard for me to imagine. So it is hard, but maybe, maybe if I had the talent, I would love to be a writer. um, I just, you know, I, I do write things from time to time and it's, it gives me a lot of fun. Um, most of the time I'm writing, but it's grants, papers of recommendation, but no writing, either writing literature, either fiction or non-fiction or a combination of, of, of both. Um, I, I, I think that would be the one thing that I can think of if I could do it right. That I, I, I could see myself do it.

Eva Amsen (26:25):
Well, you could always try it as a hobby, as we heard from Joachim Frank last time.

Eva Nogales (26:30):
Yeah, yeah. Joachim is, is very, you know, um, he's like a Renaissance man. He's a great photographer. He can write poetry. Uh, yeah. I'm not that talented, I think. Yeah.

Eva Amsen (26:45):
Well, before we, um, we end this episode, uh, do you have any advice for researchers who are just starting out in their career?

Eva Nogales (26:54):
Right. Um, I don't have one. I don't have one big idea or, or advice that will, you know, make things easier or anything like that. But, um, just, just to make them aware that if they're not already that things in science go up and down mm-hmm , and that sometimes they're going to self doubt, or they're gonna, they're gonna think that they're, they're gonna feel frustrated. They're not, they're not good enough because things don't work out. And as I heard it saying in a much more eloquent way that I, I, I could do, um, my, my friend, Carlos Bustamante said, you know, this is the nature of what we do in science, because we are constantly pushing the barrier of what is possible and what is known, uh, asking questions for which the technology that we have around is barely capable of doing it. And if you're not doing that, you're not doing science that really worthwhile doing, but it means that there's gonna be frustration. So, you know, just be aware that this happens and that it is not, you are, that are incompetent, but that you are trying to do something that is very, that is difficult. And once you do it, it will have a real meaning and otherwise surround yourself by people, you are happy working with both in your lab, your students, your postdocs, but also your colleagues, because going through those ups and downs, it is very important to be surrounded by people that you feel you can trust. Uh, you feel comfortable. You want with, you want to go and see them every morning and, and you can share the good times and the bad times get good ideas. Get good advice that is very important at the end of the day is always the human factor is your colleagues, your students, your postdocs. So that's just pay more attention to how you choose people than, or at least as much as how you choose, um, you know, your project or your technique or anything like that. That is really very important. It's impossible to emphasize it enough.

Eva Amsen (29:07):
That's good advice. So remember that it's also about people ,

Eva Nogales (29:10):
Um, oh yeah, very much so.

Eva Amsen (29:12):
Um, well thank you Ava, for joining us today. And that brings us to the end of this episode of Cryo-Talk. So thank you everyone for listening.

Eva Nogales (29:23):
Thank you. Bye-bye.

intro/outro (29:26):
Thank you for listening to Cryo-Talk a Bitesizebio podcast, sponsored by Thermo Fisher Scientific to view all audio and video recordings from this series, please visit bitesizebio.com/cryo-talk.