Molecules in motion
Why Cryo-EM, or speed-freezing molecules to create 3D images that can diagnose human cell behaviour, is taking the structural biology world by storm
CHRIS HATZIS
Eavesdrop on Experts, a podcast about stories of inspiration and insights. It’s where expert types obsess, confess and profess. I’m Chris Hatzis, let’s eavesdrop on experts changing the world - one lecture, one experiment, one interview at a time.
At school she snubbed biology in favour of physics, and years later went on to win an award for discovering how microscopic tubes work inside our biological cells. This understanding of cells at the molecular level is now paving the way for new approaches to disease management and improving the lives of humans everywhere.
Professor Eva Nogales is a biophysicist at the Lawrence Berkeley National Laboratory. She is a faculty member in the Division of Biochemistry, Biophysics and Structural Biology of the Department of Molecular and Cell Biology at the University of California, Berkeley, and a Howard Hughes Medical Institute investigator.
Professor Nogales is one of the foremost exponents of single particle cryo-electron microscopy, cryo-EM, a technique that is taking the world of structural biology by storm. She recently visited the University of Melbourne to receive the 2019 Grimwade Medal and to deliver the oration titled “Visualizing the molecular dance at the heart of human gene expression.”
Professor Eva Nogales started her career in a time where barely any women were seen in science departments. She joins Steve Grimwade to talk about her work and to look back at her illustrious career.
STEVE GRIMWADE
Professor Nogales, welcome to Eavesdrop on Experts.
EVA NOGALES
Thank you, I am delighted to be here.
STEVE GRIMWADE
Now, before we dive into your work, I thought we'd do a refresher for those, perhaps like me, who haven't thought about biology since they were in high school.
EVA NOGALES
Mm-hm.
STEVE GRIMWADE
Apologies. Basic Googling reminds me that cells are fundamental building blocks of our lives. They give us structure physically, they're stuff that I can slap, they convert nutrients into energy and they house and enable hereditary material. Look, I've tried to summarise this, but how are our basic cells working and what are they doing?
EVA NOGALES
First of all, your middle school or high school is really good. You made a fantastic summary of what cells are about. So, I actually did not study biology at college, just a little note. I was a physicist for a while. I relied also on the biology that I learned at high school. But what I do, which is actually biophysics, is to use physical methods, which in my case are very powerful electron, huge electron microscopes and then computational methods to study the fundamentals of the units of the cells. I love the fact that you said cells are things that you can touch that are a physical entity because one of the things that I study have to do with what is called the cytoskeleton of the cell. Cells like us have a structure that gives them shape and that also serve as the freeways where things move from one side to the other, so cells are very, very highly organised. They're fluid but very highly organised.
So, part of the cytoskeleton is made of a protein that's called tubulin and is called tubulin because it makes microtubules. Microtubules are little tubes that are very, very small, so there's thousands of them in every cell. They're organised like a bouquet from a point that is spread from the centre of the cell to the outside and there are other proteins that are called motor proteins. They use energy. The one that you were talking about, you said cells generate energy and then use it for whatever to keep alive and to form part of our tissues, our heart, our brains and our neurons. That part of the energy that they make is utilised to organise themselves, to give order. As a physicist I know that order always requires a source of energy. Chaos doesn't require anything. You just let things go and they become chaotic.
Cells are highly organised and they have these motor proteins that move things around like in a freeway, like the lorries in the freeway, moving things from one place to another where they are needed. Microtubules define the path at which these things are going. All of this information comes from biophysical techniques that had allowed us to look inside the cell but also extract the components that are inside the cell, put them in a test tube and then look at them with exquisite detail. We take microtubules out of the cell, we look at them in electron microscopes and we get atomic detail, so we end up knowing the chemistry of how they interact with these motor proteins. What allows the motor protein to bind and then take a step and move forward, how the microtubules themselves are built, how they are broken apart.
I am a fundamental biologist. I like to understand how cells work, but every biologist always, every molecular biologist, always makes a connection with human health. It's very obvious, right, so this is my connection with human health. Microtubules are a major target of anti-cancer agents. Why? Because one of the functions of microtubules, the organisers in the cell, is that when a cell divides the first thing that it does is it duplicates its genome, right, because then the cell is going to split in two and each one of the daughter cells have to have the whole blueprint to start anew and making everything again. It's very important that the chromosomes get split between the two daughter cells. That requires a lot of organising them and then pulling them apart. That is done by microtubules.
If you fiddle with microtubules, if you bind something to them, a small molecule, a drug, that stops the normal behaviour, the cell cannot divide. What cells are dividing the most in our bodies and in an uncontrolled fashion? Cancer cells. One of the things that I study is how drugs that act on microtubules affect them, where they bind, how could we improve them, how could we maybe make them more specific, but from a very… you know, I don't do the drug development. I provide the pharmaceutical companies, the biotech, with the basic information that they can use so that they understand how the cell works and then they can try to manipulate it to cure disease or infection or whatever it is.
That's what molecular biologists do and the kind of things that we are interested in. Understanding how the cell works at the molecular level and this is something that has repercussions ultimately for disease and for making our lives better hopefully.
STEVE GRIMWADE
Listeners, in the next episode you will be moving on to Year Two of biology at university. A fantastic summary and this brings me to this question. You've very elegantly spoken about the way things are and yet the work you do is incredibly visual. I'm interested in maybe what cryo-electron microscopy gives you. It obviously gives you an image in 2D and then that can be turned into a 3D image of the microtubules. Then what does that enable?
EVA NOGALES
Electron microscopes have been around for a long time. They are great tools not only to study biology, also to study materials like inorganic samples. It is used a lot in the computational industry because you need to check the silicon chips that are going to go into and the quality of the material and whether they're pure or not and electron microscopy can give you atomic detail. The problem with image in biological samples with an electron microscope is that when electrons go through a biological molecule like a protein or a DNA, they break to pieces. They destroy it. They make it explode. For a long time it was thought that biology, you know, that it was not going to be possible to see biological molecules in the atomic details that you can see a silicon wafer because they will be destroyed before you gather the information, but the trick is that you can cool them down. That's the cryo part.
STEVE GRIMWADE
I love this, the cooling is to about minus 200 degrees or something.
EVA NOGALES
Yeah, it's very close to absolute zero.
STEVE GRIMWADE
It's not a windy day in Melbourne.
EVA NOGALES
Well I don't know, I'm here in the summer, so I don't know how cold it gets. You're close to the Antarctica. But in any case, the trick is not only to cool them as cool as, you know, really cold, but to do it very fast. Why? So, you know that proteins are super happy and DNA are biological molecules in water. They're aqueous molecules. In water the H2O molecules are very fluid and they don't have a specific arrangement. If you cool water very slowly it makes ice. Ice is a crystal. The molecules are organised in a certain style of way and that is incompatible with the way proteins and DNA interacts with water. The trick is you freeze the water very quickly, so quickly that the water molecules don't even have time to reorganise into the crystal. That is hundreds of thousands of degrees per second.
Then you end up with this vitreous, amorphous from water that has this structure of liquid water but is in a solid. It doesn't move anymore. Because it doesn't move anymore when the electrons come, hit the protein and damage it by breaking bonds and generating radicals, but molecules that will break all the bonds like a cascade of reaction, they can't move very much because the water is solid. Because of that you have a little window that now you can collect data that has enough signals so your images have enough electrons that have gone through the sample, that you have enough [contrast] if you want that those images that are two dimensional and are still kind of noisy, like when you take a picture the flash doesn't come out and there's not enough light in the room, but we take those images of the molecules and we have millions of copies of the molecules that we extracted from the cell. Then we use computational tools, very powerful, to align those images, combine them and generate a three-dimensional representation of the molecule that is like a - you could make a clay shape if you want.
These are very complicated parts that can be interpreted as the chain of the polypeptide that make a protein and you ultimately, like in a three-dimensional parcel, thread the polypeptide, the non-sequence of the protein, the sequence of amino acid into that three-dimensional volume. At the end you have a chemical representation of where every atom is in the protein. Which part of the protein is going to be interacting with another? Which part of the protein is going to be binding DNA and opening it and copying one strand to a messenger RNA and then another? All of that information is now in that chemical representation that is a physical and chemical object. So ultimately understanding these biological molecules at truly mechanistic levels so that you could imagine in a few years we could actually make something. We understand it to a way that we could engineer it or make it up ourselves. That kind of knowledge is what we want to gain.
STEVE GRIMWADE
How long ago did your work in this begin and were you actually able to get the pictures and the images that you need now and what was happening before? So, what has changed for the people around you who need this data, who need these tools?
EVA NOGALES
The tools that I've been using have been around - they were starting to be developed around the late seventies, early eighties. The field was progressing very slowly, so always kind of breaking technical barriers, getting better microscopes, understanding better how to treat the images, how to prepare the freezing of the samples and all of that. The field was growing steadily and then something happened. There was like a singularity. One technical development that revolutionised everything and that happened about five years ago and it has to do with the detector technology. Every time we think of an imaging technique maybe we think of the microscope but at the end you have to record the image. In the old days, an electron microscope, would record the images on film, so at the end of a day at the microscope you will go into a dark room, go to the developer, the fixer, all of that process and then you will have to scan the micrograph in a digitiser so that you generate now a digital file that you can use for computer analysis.
That was tedious. There was room for a lot of human error and typically there was a cassette of about 50 images that you could take and then you have to get them out of the vacuum of the microscope. So, we needed high throughput, efficiency, this is the world we live in. So, CCD cameras, which are similar to the ones that you have in your phone, in your iPhone for taking pictures, were developed for the electron microscope where the problem is, you know, every time I talk about the electron microscope these are electrons that are moving close to the speed of light. They are very, very highly energetic, they destroy everything. They also destroy the detector itself. The silicon wafer, these electrons will come, they will be detected once and then they will fry the detector. What was being done, because we wanted the digital detector to be able to get many images, not 50 but 1000 every day that we're on the microscope, so the detector had a scintillator. A layer of glass where the electrons came and they were slowed down before they hit the actual detector and the image will be recorded.
In the process these electrons were bumping around in the glass and giving us images that were very terrible. The wonderful microscope, all our preparation and at the end the detector let us down. There was a development, there was an engineered solution for the problem of this damaging of the detector and suddenly we have a detector that works much better. It not only gives us images that are a much better representation of the true, of what the microscope is really seeing, but they have very fast readouts. We used to take an image, it took two seconds. The beam shone on the sample for about two seconds, they got integrated and it gave you an image. Now this is like a video where there are hundreds of images that are recorded during the full exposure which is as much as we can expose the sample but it still blows up, because we still have to be careful. This has many advantages because when you take a picture of someone and that person moves, what happens? That image is blurred, right?
To get the image in time you need a movie that is stopping, that is capturing one image after the other and then you play it, but if the person moves through in one single frame it's just a blur, you see nothing. So exactly, that's exactly the point, if things are moving when we take the image they're blurred, but if we are taking our movie and we can split it and then we align the frames, because this is motion that's not interesting. Remember, the sample is frozen, so it's not doing the movement that they will do in the cell, but what happens is that as the electrons go through the sample the sample buckles. It kind of ripples and then the image is blurred. But with this movie what we can do is correct that motion. Imagine my hand is moving but I take every frame and I align it computationally and now I have an image of my hand that has high contrast and that is not moving anymore.
Long story short, these new detectors have revolutionised what we can do. Before we were limited to poor resolution and now we are able to get atomic details. Visualise the position of the atoms. Furthermore, when you freeze your sample your molecules are truly moving. One will be like this, one will be in another position [with the arm extended, close], far away and when they're frozen there are many copies and each one is in a different position. The images are so good now that we can realise that they are in different positions, separate them and our structures describe the whole, what we call the conformational ensemble, which is a fancy way of saying we're seeing the full range of motion.
That's information that is truly useful because we refer to these to proteins as molecular machines. Machines move and when you see them moving it's when you understand what they do, so this has really revolutionised how we look at biological processes and that did not exist before.
STEVE GRIMWADE
This is now exploding. I think you recently said that now is a brilliant time to be doing this type of research. Actually, you may have said this a couple of years ago potentially. You said the technique is basically exploding right now. Everybody wants to use it. So how is this being used by others?
EVA NOGALES
It used to be that cryo-EM was a niche technique.
STEVE GRIMWADE
Cryo-EM is cryo electric…
EVA NOGALES
Sorry. Cryo-EM is cryo electron microscopy, so microscopy of frozen samples. It was a very niche technique where there were a handful of people in the world that had been training a few labs scattered here and there, that were going along and producing papers, scientific resource, at a certain rate. Now many people that are interested in how biological molecular machines work are jumping into it because, as I said, now it is much more informative, higher resolution, more information on motions, but also it has become easier. Okay. As fields evolved all the technique becomes, many aspects of the technique, becomes automated. Kind of idiot-proof. So, it is easier to jump into the field with what becomes shorter and shorter learning periods and they start producing amazing results. So now there are many laboratories across the world that are learning this technique, using facilities, regional and national facilities.
These facilities are growing. You guys have some here at Melbourne. These are people that were not experts of how an electron microscope works or how do you freeze a sample fast enough to be able to look at it in their electron microscope, that now are able to do it because of how all of these techniques have evolved to be easier to use and achieve more faster in a very efficient way.
STEVE GRIMWADE
When I think about trying to understand life you often go either really big or really small. You often go to the astrophysicists and say how does the universe work and they can give us an idea of why we exist potentially or not. Likewise, I guess you can go to the very small and go why do we exist? How is this cell working? What are you finding out for yourself? This is probably more a personal question than a scientific one.
EVA NOGALES
Mm-hm.
STEVE GRIMWADE
Or maybe it's both.
EVA NOGALES
What I find interesting and what I think is my niche within this later scale of things… can I tell you an anecdote?
STEVE GRIMWADE
Please.
EVA NOGALES
I was being given an award when I was significantly younger, but in any case…
STEVE GRIMWADE
Yeah, I told the listeners before about the three pages of honours. You've done…
EVA NOGALES
No, no, there's not, you're exaggerating. But this was when - after my postdoctoral work when I saw the structure of tubulin, the protein that makes these microtubules that I was telling you about and I actually obtained [it bound] to Taxol, which is the most broadly used anti-cancer drug for solid tumours. I was given this award and the previous year it had been given to Saul Perlmutter who was the person that discovered that the universe is expanding. It was funny because I said if the cash prize that you gave was proportional to the size Saul Perlmutter will be a millionaire and I will be giving you money back because the things that I study are very small. But I think it's a fascinating world because they're small but they're alive. It's not like people that are studying atoms. I'm studying things that at the microscopic level are actually very complicated. They have not three atoms or 1000 atoms. They have hundreds of thousands and they are alive in the cells that they consume energy, they do motion, they partner, they separate, they pull, they push, they're fascinating.
I see them as life entities in that sense that collectively give rise to life as we know it as organisms. Tissues, organisms and all the processes that I study are very fundamental so they are happening in all of our cells. They're happening in your neurons, they're happening in your skin, in the lining of your stomach, in your heart. The processes that I study are just essential and it's just something very rewarding about understanding how the complexity of life starts with these small molecules that have evolved to do very, very complicated things and keep us alive.
STEVE GRIMWADE
What I love about your work and I know just the bare surface of it is that you are not reductive or singular. There is not one area of study that you do. I think there's a lot of connections. I think about the work of the lab and I think that obviously there's theoretical science and there's your physics and there's biology, but there's also technical. There's literally the engineering behind the microscope itself and the creativity…
EVA NOGALES
Mm-hm.
STEVE GRIMWADE
…and there's a lot of things happening together. How do you bring these ideas together or what you would consider your major strength?
EVA NOGALES
I think the most important thing, to be honest, is to come out with an interesting question. What is it that we don't know yet and it will be fabulous to be able to know? Once you frame that question and how important it will be to answer it then you say, now, how do I go about answering it? Then you break the problem into pieces and say how do I solve each one? What is accessible? What is available for me? At which point I have to bridge an experimental gap because no-one has done this before. That's the way you build it up but I think the most important thing is to come up with that question and then have the resourcefulness to look around and see how can I pull from different disciplines to be able to answer this? It's very common but I would say that the twenty-first century is the century of biology. I think the twentieth century, the nineteenth century, were the centuries of physics and of chemistry. Of course physicists and chemists will argue with me, but I honestly think it is the case. There's so much that we still don't know and if you will remind me I'll tell you another anecdote in a second.
What is happening is that the problems that we are facing are so difficult that people are being drawn into biology from many different disciplines. There are many chemists that are doing what is called chemical biology, to probe biology, to answer questions, but using very clever chemical tricks. Of course physicists like me are coming in with techniques like electron microscopy to be able to visualise these molecules. Engineers are pulling in to solve those technical problems like the detector that needed to be solved. You need all of these expertise to be able to tackle the most fundamental problems and I think that's something that is very interesting about how science is done. It's done by teams, it's done by using expertise and knowledge and experience from very many. Especially when you are asking the most difficult questions that are going to give you really interesting answers.
STEVE GRIMWADE
I have a question about the way the University of Berkeley approaches this, the University of California and Berkeley, however do you want to go to your anecdote?
STEVE GRIMWADE
Impossible question to answer, but what's the next biggest mystery?
EVA NOGALES
Oh, my god. I would say it's not my field. I mean there will be components maybe that trickle down to the molecular details that I deal with, but if I have to start over right now, I think it would be system neurobiology. Someone that wants to understand memory, consciousness, learning, behaviour, but want to understand it in a rigorous way, that ultimately comes down to neuronal connections and molecules of synapses and things like that. How does this very complicated behaviour, these complicated properties that have to do with the way we think, how they come out at the basic principal from molecules themselves that are in our brain? I think that's a fascinating thing and we are not even in a square one right now but super exciting.
STEVE GRIMWADE
For you being a Spanish woman…
EVA NOGALES
Mm-hm.
STEVE GRIMWADE
…I'm guessing that you have decided that you are open to travelling the world to fulfil your work and your dreams. Do you have to be mobile to succeed?
EVA NOGALES
I think it's, you know, if you have more possibilities to choose that's always a plus. I don't think that you have to be constantly moving to achieve your goals and people do sometimes very, very good work without having to move around. I just think that when it comes to science you want to have as many doors opened as possible, but I don't think there is a set of rules of what it is that you have to do to become successful. I'm from Spain, in Spain people don't move really. I mean you live with your parents through college, graduate school. You do that until you're married and you buy a house two streets down.
STEVE GRIMWADE
You go to the university that's closest to where you live.
EVA NOGALES
Absolutely, yeah. That's a rule. The only reason why you…
STEVE GRIMWADE
That's a rule-rule. That's not a social rule.
EVA NOGALES
It's a pretty rule. It is very much a rule. I think you have the - things may have changed since. I'm not there, which it has been a while, but unless you want to study something that is not offered at the university that you're in and then you move to the next university in your city. Right. There are exceptions to that but that is very typical as part of the, I would say, Southern European style that families stay very close. Because of that your college, your work, your jobs, they tend to be very close. I'm actually the black sheep in my family because I left. I left after my undergraduate degree. I went to the UK for my PhD, had a very hard time with food there, so I moved to California where their food is exceptionally good for my postdoc and much better weather. I moved in a way just because of the opportunities that are open to me at any given time.
STEVE GRIMWADE
I'm interested. I mean your story, it seems to be one from the future because reading your background I think you were inspired by three teachers you had.
EVA NOGALES
Oh yeah.
STEVE GRIMWADE
Three female teachers I think maths, physics and biology.
EVA NOGALES
Mm-hm.
STEVE GRIMWADE
All women and leaders in their field and here you are a leader in your field. Is there any problem in getting women into STEM?
EVA NOGALES
There is. I mean and this is a very well-known issue that is getting better with time but very remarkably slowly. There are certain areas where it is very hard to get women and I don't understand why. I remember my kids, I have two boys and now they're more grown up, but I remember from day one going to daycare. The kids were two and this happens all the way until they're 18. You go into the day care centre and the girls are sitting down and making beautiful drawings, so organised and the boys are on the floor hitting their heads. You say how can it end up being the way it is? But somehow there is this idea that women are good at social sciences, literature, medical things and the guys are good at math and engineering and physics. It's just…
STEVE GRIMWADE
Hitting their heads on the floor.
EVA NOGALES
Hitting their heads on the floor and… for some reason something happened. I was very fortunate because I had these three role models during my high school, but I have to tell you, I decided to go to physics, to study physics, there wasn't a single female faculty in my department. None. It was a time of change so in my class we were already maybe, maybe a third, maybe a little bit less than a third, of the students were women, but in the faculty, there was no-one and at the graduate level there was one. It was very hard to find role models. Biology is interesting, it is kind of intermediate. Even within biology's different areas, so there's cell biology, neurobiology, there's more women. If you go into biophysics, something that is seen more as the hard sciences, more math, more computational, there's fewer. I don't know why. This is about to change.
There’s obviously many cases of extremely successful women, mathematicians, engineers, pilots, whatever. Things that have not traditionally been seen as women jobs and roles, that slowly we will conquer it. This is a process where having more role models, more people that you can see and identify, look, I look like her and she is doing this thing, that are motivational and that we will eventually get to a situation that is more equal.
STEVE GRIMWADE
Speaking of motivation, what is something you would tell your students? What is one bit of advice you would pass on?
EVA NOGALES
My god, one.
STEVE GRIMWADE
Move to California for the food. Okay, give me a second one.
EVA NOGALES
All right. No, so I always thought on this that they have to, you know, to be a scientist you have to love the question, but you also have to love the path to answering this question. This is very tough because by definition it is something that I, you know, one of my colleagues at Berkeley, Carlos Bustamante, just articulated very, very well, so I don't know whether I'm going to be able to do this as well as him. If you are a scientist you are breaking ground all the time. You are always doing an experiment that no-one has done before. There is a very high likelihood that it's not going to work the first time, the second or even the third, so you are constantly battling and facing your own limitations. You have to have some kind of inner strength and confidence in that what you're doing is, first, important and second, you will be able to do it if you persist, but you are always feeling that level of incompetence that have to do with breaking new ground.
This doesn't happen with other professions where you learn the rules and you just go do it. The life of the scientist is different every day and most of the time you're just failing and learning from your failure and then you succeed and its hooray, right. That's the most important thing is you have to have the motivation to keep going because I can assure you, you're going to fail. If you are doing anything that is interesting there is going to be a lot of failure before you succeed. That is one thing that I tell them.
STEVE GRIMWADE
Have you ever run out into the streets and thrown your papers in the air and yelled eureka?
EVA NOGALES
I don't know if - I may have done it in an office in front of a computer.
STEVE GRIMWADE
I think I watched a really bad movie once and so I can…
EVA NOGALES
Yeah, yeah. I do not like the way scientists are portrayed by Hollywood and all the movie industries. I have a…
STEVE GRIMWADE
I love those explosions in space. They are amazing and sound so great. Look, finally, next time I'm scratching myself and I'm trying to picture the microscopic world beneath my skin, what do you want me to think about?
EVA NOGALES
Oh, I want you to think about these entities that are all the way down to this microscopic level that are working very hard for you to repair your skin to build up, to move around cells that cover them, but that at the atomic level and at this little, I don't know, not automatons, but a moving organism consuming energy that they get from the hamburger you had earlier in the day to do that work, to keep you alive, to keep you regenerating and breathing and walking around.
STEVE GRIMWADE
Professor Eva Nogales, thank you so much for joining us today.
EVA NOGALES
It was a pleasure.
CHRIS HATZIS
Thank you to Eva Nogales, biophysicist at the Lawrence Berkeley National Laboratory, University of California, Berkeley. And thanks to our reporter Steve Grimwade.
Eavesdrop on Experts - stories of inspiration and insights - was made possible by the University of Melbourne. This episode was recorded on February 14, 2019. You’ll find a full transcript on the Pursuit website. Audio engineering by me, Chris Hatzis. Co-production - Silvi Vann-Wall and Dr Andi Horvath. Eavesdrop on Experts is licensed under Creative Commons, Copyright 2019, The University of Melbourne. Don’t forget to drop us a review on iTunes, and check out the rest of the Eavesdrop episodes in our archive. I’m Chris Hatzis, producer and editor. Join us again next time for another Eavesdrop on Experts.
Professor Eva Nogales started her career in a time where barely any women were seen in science departments. In college, she skipped biology to focus on physics, relying on her high-school knowledge of the former to shape her career as a biophysicist.
Now, she’s using her understanding of the microtubules in our cells for improving disease management, including slowing the uncontrollable growth of cancer.
This niche understanding of our cell behaviour at the molecular level is already improving the lives of humans everywhere, and the technique used by Professor Nogales called “cryo-EM” is taking the world of structural biology by storm.
She recently visited the University of Melbourne to receive the 2019 Grimwade Medal, and to deliver the oration titled: Visualising the molecular dance at the heart of human gene expression.
Episode recorded: February 14, 2019
Interviewer: Steve Grimwade
Producer and editor: Chris Hatzis
Co-production: Silvi Vann-Wall and Dr Andi Horvath
Banner: Berkeley Lab
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