Dr. Susan Hafenstein, The Hormel Institute’s First Cryo-EM Core Program Director, Explores Uncharted Territory at the Microscopic Level
Dr. Hafenstein stepped to the helm as The Hormel Institute’s first Director of the Cryo-Electron Microscopy (Cryo-EM) core program and Professor of Biochemistry, Molecular Biology, and Biophysics in August. Most recently, Dr. Hafenstein served as Penn State University’s Director for the Center of Structural Biology, Huck Chair of Structural Virology, and Professor of Biochemistry and Molecular Biology. Dr. Hafenstein’s hiring marks a new phase for The Hormel Institute as she joins our robust team of researchers to embark on new territory in establishing the Minnesota Bioimaging Center (MBiC), a project which involves expanding The Hormel Institute’s current cryo-EM and cryo-ET capabilities to be of greater use to researchers within the Institute and across the Midwest region. “I really like doing that [helping other researchers use cryo-EM]. There’s already four people here doing cryo-EM,” Dr. Hafenstein said. “The mission is to create a facility that all of Minnesota can use—and even more than that, the Midwest.” Having been on board at The Hormel Institute for just a couple weeks, the setup of her lab is still taking shape. Boxes have slowly piled up, their contents ready to be unboxed again. The lights are on a little more frequently now. New faces frequent the expansive halls. A laser-printed cardboard cutout hangs in one of her lab windows. It’s a 3D rendering of HPV, somewhat similar in shape to a macrame soccer ball, in varied hues of blue to show its intricate depth and texture. Move in closer to the picture, and you’ll likely notice a few new layers of threads you hadn’t before. The cutout itself is a point of pride: Susan’s lab was the first to render a high-resolution image of the HPV virus’s structure. She likes the idea of adding other viruses to the display in the future, which could illustrate just how different in scale virus sizes can be. Compared to this HPV that’s about 26 inches in diameter, a herpes virus would stand nearly 8 feet tall, for example. Varied, too, are the paths a scientist can take. Here’s what work as a scientist has looked like for Susan—and what she thinks it takes to become one.
Setting course
Dr. Hafenstein says she’s known she wanted to be a scientist since childhood. “Ever since I was a kid, I wanted to understand how things worked,” Susan said. She was also fascinated by the idea of exploration—enthralled by tales of Jack London, for example, and stories about early explorers and their travels to remote corners of the globe, like Antarctica. “I either wanted to discover a new continent or a new antibiotic—so there was that love of discovery,” she laughed. And there were some things in particular that fanned the flames of her initial curiosity for the sciences and that desire to explore the unknown—such as the book Microbe Hunters by Paul de Kruif, in which each chapter dramatizes discoveries related to particular microbes and medical breakthroughs that occurred because of them, as well as the people behind them, such as Pasteur or Fleming. “There were different chapters for different scientists … all these early leaders who made these discoveries that affected everything: the discovery of antibiotics, sterile technique … I think that book was a turning point for me. I was like, ‘I wanna do that!’” Susan doesn’t think she’s alone in that. “I think a lot of scientists have that,” Susan said. “It’s exciting to be the first person to visualize something that no one else has seen, or figure something out, how it works. Especially if it’s something we can use to our advantage as humans.”
Map maker to map solver
Since Earth’s continents were pretty well discovered by Susan’s time, she ultimately set off to explore the microcosmos, where she now collaborates with fellow researchers to map microscopic structures such as viruses. That’s included viruses like HPV, murine papillomavirus, polyomavirus, polio virus, canine parvovirus, and Zika virus, to name just a few. In much of Susan’s work, one of the primary goals of studying viral structures is to learn more about how they function—especially in terms of how they interact with host cells to gain entry, which is how infections begin. Making these viral maps—or rather, “solving” maps, as one might say in her line of work—can eventually help scientists use aspects of a harmful virus’s structure against itself to either prevent infections or treat patients more effectively after infection has occurred. “It’s kind of satisfying to be able to solve these problems one at a time and succeed with a high-resolution structure,” said Susan. “...Once you have that structure, you have a new tool that you can use to basically defeat the virus itself. I mean, that sounds silly: ‘We get the structure of the virus and then we use it [its own structure] against it’—but that’s exactly what we do.”
What is cryo-EM?
Cryo-EM, short for cryogenic-sample electron microscopy, is an essential tool that makes map-solving at this level possible—but they’re few in number across the country. Fortunately, The Hormel Institute is one of the select few places where the technology is currently available. “Here [at The Hormel Institute] we have a state-of-the-art cryo-EM microscope [the Titan Krios] … with all of the add-ons that you can get right now—so it’s the best of everything that’s available,” said Dr. Hafenstein. A variety of tools and techniques can be used in combination with a cryo-EM microscope, but generally speaking, the process involves flash-freezing numerous iterations of the desired sample—such as a virus, protein, or cell—then inserting it into the microscope for analysis. Inside the microscope, an electron beam is fired at the frozen sheet of sample, which will cast “shadows” or shapes onto a sensor below. That information can then be analyzed by specialized software to group together similar images of the sample from a particular angle for analysis. With enough of these composite images, it’s then possible to group these images together to render a 3D structural map of a sample. Under the right conditions, scientists can develop renderings of a sample with atomic-level resolution. Beyond these basics, there are two main approaches one can take when using cryo-EM: one for high-resolution, single-particle analysis, and another for low-resolution tomography. The key difference between the two is that with tomography, the scientist will begin tilting the sample grid as images are captured. But because the stage can only be tilted so far for logistical reasons—about 60 degrees—the images are poorer in resolution compared to the ones that can be generated by single-particle imaging. Still, gathering enough of these angles is what makes the 3D renderings of some subjects possible, says Dr. Hafenstein. “It’s [tomography is] still extremely powerful, because we can see pleomorphic things [that is, things that can alter their size or shape under different environmental conditions] instead of all the same thing that we can average altogether . Now we can see things like HIV, or coronavirus—because each one is different. Otherwise, we wouldn’t get a look at it.”
Solving maps to solve viral problems
Dr. Hafenstein’s lab at Penn State University was able to develop the first high-resolution maps of HPV as a 3D structure using cryo-EM. And cryo-EM remains essential for the Hafenstein lab at The Hormel Institute as they seek to solve the structural map of one of HPV’s proteins known as L2. HPV’s capsid (its shell) consists of two proteins called L1 and L2. Dr. Hafenstein’s team has enough data to know the sequence of L2, the minor capsid protein, is there—and it has the potential to be a better candidate to target for prevention, as it appears to play a crucial role in helping the virus enter human cells. “It [cryo-EM] gives us a better tool to try to find the minor structure protein to try and understand mechanisms of entry, how does it get inside the human host, which is the first step to causing cancer: it has to get in,” said Dr. Hafenstein. One of the hopes is that with better understanding of L2, scientists can develop a way to prevent the virus from binding to human cells—especially since the current range of HPV vaccine options available to patients only protect against a certain selection of all HPV types. Since L2 is conserved across all the serotypes (distinct groups within a particular species of microorganism) of HPV that cause cancer, a vaccine targeting the L2 protein has the potential to protect against each one. “If we could design a vaccine to this lesser protein, it could give us broader [HPV] protection,” said Dr. Hafenstein. But it’s a task akin to trying to find a needle in a haystack. Fortunately, coupling cryo-EM with other techniques and specialized software can help to more quickly identify key areas where the elusive L2 may be hiding. “We have the virus purified itself all the way to 1.9Å, which is atomic resolution,” said Dr. Hafenstein. “And this is very exciting, because all the atomic details that have been hidden, we can now see.” Å, the symbol for “angstrom,” is a unit of measurement commonly used at the microscopic level. Dr. Hafenstein says most experts would agree that 2.2Å or lower would achieve atomic resolution—a feat some microscopists occasionally call “breaking two.” “You can see all the side chains and how the proteins interact with each other to make the capsid. … You can see water molecules, we can see post-translational modifications, I mean, it’s all there,” she said. And somewhere in there, the L2 protein awaits discovery—and a structural map of its own. The team has enough information to know it’s located inside a pore of the capsid, and that parts of L2 protrude from the capsid, where it binds to antibodies that can neutralize it. But with the atomic resolution map generated using cryo-EM, plus some additional tools and techniques like specialized software and tomography, they can narrow down its possible whereabouts further. “So now we’re using a combination of techniques to get this sort of picture: that we have the capsid, it’s protected to about [a certain point], and we know these pieces are outside the capsid. … Once we identify for sure which ones are outside, those will tell us the targets that will make good vaccines,” said Dr. Hafenstein.
The art of the science
Beyond the joy for discovery, Susan supposes successful scientists must have something more: a love for the science itself. “Because if you don’t have perseverance, and just sheer love, when things aren’t working well, you’re going to start getting down. So there has to be some sort of underlying enthusiasm, or you’re going to run out of gas at some point,” she said. For persistent scientists in this ever-advancing field, there’s plenty of room for experimentation while in pursuit of discovery. “There’s still a lot more art than science in some ways when it comes to preparing the cryo-EM sample,” Dr. Hafenstein said. There are three main components of cryo-EM, a trifecta of sorts, each of which can have a major impact on the quality of sample images:
- Sample preparation
- Data collection
- Data processing
Collaborating with colleagues to swap cryo-EM tips and tricks for how they approach generating images of samples is one aspect of the role as Director of the Cryo-EM core program that Dr. Hafenstein is particularly excited about. Dr. Hafenstein sees plenty more opportunity for advancement in primarily two of the three key areas: sample preparation and software development. Refining approaches in these areas could help scientists achieve the highest possible resolution when capturing sample images. In sample preparation, one recurring question, particularly for single-particle imaging, is: “How can we work toward in situ?” In microscopy, “in situ” refers to observing a sample in a way that allows one to “catch it in the act” of a particular interaction or process. The Hafenstein lab has been experimenting with this by using nanodiscs or liposomes to immobilize receptors the virus would recognize. Then, they can vitrify the sample to create “freeze frames” of the virus interacting with a cell membrane at these receptor points—and at a higher resolution—to learn more. Then, there’s the matter of structure and material choice when it comes to grids. For example, carbon is a common grid material, but some of its properties can present challenges when analyzing certain types of samples. Dr. Hafenstein plans to collaborate with other University of Minnesota colleagues to develop gold hexafoil grids, since gold is much stabler by comparison. When it comes to software, there are plenty more opportunities for tweaks. For example, one could make software adjustments to better visualize a virus structure’s asymmetry. The Hafenstein lab did just that with HPV. “Viruses have all this lovely symmetry—but when it’s interacting with the host, it sort of breaks down. That’s what this is,” Dr. Hafenstein said, gesturing to two styrofoam icosahedrons on her desk. One has multicolored push pins inserted haphazardly all over its surface; the other does not. “So this virus [the one with pins] is asymmetric now. Because it’s only bound in some places, and some places remain unbound by host proteins. “In order to see that, we had to develop special software. There’s some software that’s similar out there in the world,” said Dr. Hafenstein, laughing. “We did a homemade version that allows us to store where on the capsid each protein is bound.”
The fairest of them all
Susan’s enthusiasm for her work is palpable, contagious—dare we say, viral? It’s easy to understand why. The sizes and structures of viruses framed along her office wall, or 3D-printed models on her office shelves, show just how diverse they can be in size and appearance—and ultimately, how they function. Each one presents itself as its own abstract sculpture. When asked if she has a favorite structure in particular that she’s studied with cryo-EM, her long pause shows just how hard it is for her to choose. “Wow—that’s a tough question to answer.” Another period of silence. “All of them,” she says, laughing again. “Every discovery is exciting in its own way.” While it’s hard to settle on just one, there are still plenty that stand out. Like her first solo structure. Susan gestures to a framed journal cover featuring a sapphire sphere coated in what looks like macaroni noodles. The virus is Coxsackievirus B3, and the “macaroni noodles” are a protein known as decay-accelerating factor (DAF): “My first structure, we learned so much. A single change in the capsid can change the binding of the host protein,” Susan said. Parvovirus B19, a parvovirus that can infect humans, also stands out for the discoveries that came with it. “We found these host proteins bound to it [B19] from human serum. I mean, it circulates in the blood, and it has adapted to coat itself with human proteins—probably to hide from the immune system.” Then there’s HPV “in all its 1.9 angstrom glory,” as Susan says. “I mean, it’s gorgeous. And it’s so exciting to be able to see every single detail.” Not that she’s a fan of the viruses themselves, she clarifies. After all, the reason she and her team members are investigating them at all is to identify potential ways to eradicate them. “All my children—I love all my children. I don’t love any of my children more than the others,” Susan insists, laughing.
Science as service
Susan believes another important aspect in the work of being a scientist is the role of service: to those who may directly or indirectly benefit from the research at hand—as well as to when it comes to collaboration with and mentorship of fellow scientists. The service aspect is yet another one of the factors that excites her about her role at The Hormel Institute. There are opportunities here to use cryo-EM and other specialized equipment for her own research—but there are also opportunities for collaboration, including with other virologists, and to help others make the best possible use of the tools The HI has at hand. She recalled the important role her mentor in her PhD program at University of Arizona, Ben Fang, played in her professional development—and how he displayed what it looked like to be a mentor to others. “He could teach a subject at every level. ... He could meet you on any level and answer your question in a very respectful and interesting way,” she said. It was that mentorship, among other factors, that she thinks directed her into the area of research she conducts today. “But it was the viruses, too,” she laughs. She aims to carry that same sort of leadership style in her own lab. “The people in my lab, they’re the next generation,” Susan said. It should be noted that Dr. Hafenstein didn't make the cross-country trek to Minnesota alone. Seven of her colleagues who worked in Hafenstein’s lab at Penn State University now join the Hormel Institute to continue their work. “They’re an excellent group,” Dr. Hafenstein said. “…Each one has a certain level of expertise, we’re sort of stair-stepped, and so the people who know more are in the same room with the people who are learning. … Everybody has a helpful attitude, where they want to see the other person succeed. My lab excels at that.”
Full steam ahead
Moving forward, Dr. Hafenstein’s primary goals are threefold:
- Accomplishing goals outlined by The Hormel Insitute’s MBiC project
- Expanding tomography capabilities at The Hormel Institute
- Helping scientists get the highest quality possible when capturing images of samples for their research
In a field so rife with opportunity, and at a research facility set to expand its current cryo-EM capabilities, a few words from Dr. Hafenstein sum things up well: “We have lots to do.”