Cover Story
A Cold, Hard Look at Macromolecules
Cryo-EM, an advanced microscopy technique that utilizes extremely cold temperatures and electron beams to illuminate the structures of some of the tiniest building blocks of life, has come to UW after years of investment — and it could help CALS scientists reach new frontiers in the biosciences
At its most basic level, it takes pictures. For biochemistry professor Elizabeth Wright, that’s the scaled-down explanation of cryogenic electron microscopy, or cryo-EM. But it’s so much more than that. Through rapid freezing, controlled beams, and advanced lenses, this game-changing research tool reveals the intricate architecture of cells, viruses, and proteins, all at molecular resolution — or better. Cryo-EM makes the truly complex much easier to understand.
“It’s satisfying to think, both creatively and technically, about how to take images that reveal information at a structural level about a particular object,” says Wright, who is also an affiliate with UW’s Morgridge Institute for Research and a leader of cryo-EM efforts on campus. “One thing that makes life fun in my line of work is being among the first people to see something and to be able to provide scientists and others an understanding and appreciation for what we are seeing.”
Cryo-EM is revolutionizing the biosciences by enabling imaging of macromolecules, viruses, and cellular substructures at near-atomic to atomic resolutions. Advances in detectors, computation, and electron microscopy design now produce high-resolution images of rapidly fro- zen samples that rival current methods, such as X-ray crystallography, because these new techniques require far less material, capture natural states, do not require crystallization, and allow the advantages of high-contrast, time-resolved imaging.
With cryo-EM, scientists can peer into the surfaces where drugs and proteins interact, where diseases occur, and where viruses orchestrate their attacks. It has the potential to impact every corner of medicine, from Alzheimer’s research to vaccine development to protein and cellular engineering. And its reach extends to many other research areas, including biofuels, engineering, and computer sciences.
Scientists from CALS and throughout UW have already been using cryo-EM to make advancements in these fields, but they’ve had to rely on facilities outside of the university. Now they’ll have a place right on campus where they can conduct their groundbreaking work. The UW–Madison Cryo-EM Research Center (CEMRC) is set to open in spring 2021, and the Midwest Center for Cryo Electron Tomography (MCCET) will soon follow with an opening in early 2022.
Both centers, to be housed in the Hector F. DeLuca Biochemical Sciences Complex, will be directed by Wright. And they both represent a continuation of the university’s long history of contributions to structural and cell biology, virology, and medicine, as well as a major return on long-term campus investment in the technology (see sidebar). The centers will be pivotal in many ways: for building on the important work of talented researchers of the past and present, for honing UW’s competitive edge in a rapidly evolving field, and for making vital discoveries that have the potential to transform lives.
Sidebar: How Does Cryo-EM Work?
During the cryo-EM process, aqueous samples are frozen, or vitrified, at approximately -183 °C (about -297 °F). The rapid freezing process supports the formation of a non-crystalline phase of ice that is amorphous and vitreous (glass-like). No dyes or other alterations are needed to view the structures.
Next, electron beams are shot at the frozen molecules in the microscope to capture an image of their structure. When electrons hit the biological sample in the microscope, they scatter and then pass through a series of lenses to generate an image. In single particle cryo-EM, which is often used on protein complexes, hundreds of thousands of such images in random orientations are combined digitally to reconstruct the molecular structure.
Central to Bioscience Success
The goal of the CEMRC is to provide instrumentation, technical assistance, training, and access to cryo-EM for the UW–Madison research community.
The facility houses four Thermo Scientific microscopes in three buildings, including the powerful Titan Krios G3i cryo-transmission electron microscope (TEM), located on the first floor of the Biochemical Sciences Building. A smaller Talos TEM, which is the first step in the cryo-EM research pipeline, is located in the Biophysical Instrumentation Facility and Biochemistry Optical Core. The Deluca Biochemistry Building houses two more state-of-the-art microscopes — a Talos Arctica cryo-TEM and an Aquilos cryo-FIB-SEM — as well as specimen prep equipment and lab space for UW investigators, external collaborators, and industry partners.
Construction and installation have been complex because the rooms need tight temperature and humidity control and sophisticated shielding to make sure the microscopes are acoustically, electronically, and vibrationally isolated. All of this advanced technology requires highly trained personnel. Staff have been busy preparing to support the center’s users. In 2019, Eric Montemayor, CEMRC facility manager, served for three months as an embedded trainee at the National Center for Cryo-EM Access and Training (NCCAT), which is housed in the New York Structural Biology Center at The City College of New York and is funded by the National Institutes of Health (NIH). CEMRC systems administrator Matt Larson, a computer scientist with a doctoral degree in physiology and biophysics, has also been essential to the launch, and he will continue to be so as he develops new computational systems for the center.
UW researchers are planning to use the center in diverse and innovative ways that will help keep the campus at the bioscience frontier. Word is spreading about the CEMRC, laying the groundwork for collaborations.
“Some of our researchers understand how to do the computational aspects of the pipeline, and we may just support them with sample optimization and cryo-preservation and data acquisition and then hand off images where they handle the computations on their own,” Wright says. “For other investigators, we support them through the entire process and provide them with their structure and its interpretation on the back end.”
A recent masked and physically distanced tour of the CEMRC was a simultaneous walk down memory lane and glimpse into the future for Kate VandenBosch, dean of CALS, and Bill Barker, associate dean for research. Both used electron microscopy earlier in their research careers. For VandenBosch, it was a treat to get up close and personal with technological advances she says she could only have dreamed of in her postdoc days, and it enhanced her appreciation of the privilege that comes with peering into the inner workings of life.
“With EM, I always felt that I had a window into the beauty of biological structure that most people didn’t get to see,” she says.
Barker couldn’t help but imagine what cryo-EM capabilities will bring to CALS. “Now it will be possible to explore the nature of soil organic matter and microbial communities in a hydrated state, or the structure of the rhizosphere, or the spike protein of the novel coronavirus,” he says. “And just imagine what this technology could add to our understanding of frozen dairy desserts!”
As wowed as they were by the array of brand new, state-of-the-art equipment, VandenBosch and Barker were more impressed by the vision and hard work of the people.
“It was equally exciting for me to meet today’s postdocs and staff who are putting the ‘scopes through their paces, making sure things are running as they should, and perfecting specimen preparation,” VandenBosch says. “The machines are impressive, but it’s the human element that will really make this facility soar. Their expertise will enable other users to ask and answer important biological questions.”
But the CEMRC is only one of two cryo-EM facilities set to open at UW. Construction of a national hub on campus is also underway, in partnership with the UW Division of Facilities Planning & Management.
In 2019, NIH announced it was investing in cryo-electron tomography (cryo-ET) to get ahead of the game in understanding how cells work and respond to viral and bacterial infection, neurodegeneration, and more. In September 2020, the NIH announced that it will provide $22.7 million over six years to create a national research and training hub at UW–Madison — the MCCET. The center will support investigators by providing access to well-trained staff and state-of-the-art equipment for routine and advanced cryo-ET specimen preparation, data collection, and computation. The MCCET will also provide hands-on, remote, and virtual training in cryo-ET specimen preparation, data collection, and data processing and validation.
MCCET staff will collaborate with centers at the University of Colorado Boulder, the New York Structural Biology Center, and the SLAC National Accelerator Laboratory to offer the research community cryo-ET training and access. The NIH is supporting the large investments needed to pay for the equipment, personnel, and service contracts necessary to operate these cutting-edge research facilities.
Members of Wright’s team will have roles at both the national hub and the UW center. It will take a year to renovate the space for the national hub, order the equipment, and complete its installation, so it is scheduled to be fully operational by early 2022.
“Often, in structural biology, we work as separate units, and having this network of centers is special because we are building a community,” Wright says. “This allows us to work as a larger team of cryo-EM pioneers to support the greater research community. Each one of the new cryo-ET centers has its own strengths and specialization in how staff consider processing samples and data collection.”
The UW centers also create jobs and are leading to engagements with surrounding tech and biotech companies. The CEMRC and UW–Madison are pursuing non-disclosure and confidential disclosure agreements with companies that are developing new drugs and therapeutics.
“We look forward to long-term partnerships with these companies,” Wright says. “We are also using training grants to provide internships for our students to bring their advanced training to industry. We can be a nucleating point to do a lot of good for the state and bring people together.”
A Lure for the A-List
UW–Madison is also making major investments in the people who rely on cryo-EM and cryo-ET for their research. Wright’s experience with both technologies was critical to securing the NIH hub.
Wright completed her undergraduate education in biology and chemistry at Columbus State University and her Ph.D. in chemistry at Emory University, followed by postdoctoral work at the University of Southern California and the California Institute of Technology. She was an associate professor in the Department of Pediatrics at the Emory University School of Medicine and the director of the Robert P. Apkarian Integrated Electron Microscopy Core at Emory University before joining UW–Madison in 2018.
Wright studies pathogenic bacteria and how cells regulate interactions with the environment. She also investigates the structures of viruses such as HIV-1, measles virus, and respiratory syncytial virus, and she explores neurodegenerative diseases, such as Alzheimer’s, caused by defective proteins. Understanding at the molecular level how these proteins impact neuronal cell structure and function can help in the development of new therapeutic and curative approaches.
The large neuroscience group and Alzheimer’s Disease Research Center on campus were important factors in Wright’s decision to call UW–Madison home. The same is true of several other recent additions to the faculty.
The Department of Biochemistry and CALS have also hired assistant professors Robert Kirchdoerfer BS’06, an expert in cryo-EM applications in virology, and Ci Ji Lim, an authority in cryo-EM applications in DNA biology. The Morgridge Institute for Research hired investigator and assistant professor of biochemistry Tim Grant, who develops computer programs to improve cryo-EM and cryo-ET data collection and analysis, and investigator Brian Bockelman, who specializes in research computation.
When Kirchdoerfer was offered a faculty position in the Department of Biochemistry and Institute for Molecular Virology in 2019, it was a homecoming. The Oregon, Wisconsin native was hired as part of the Metastructures of Viral Infection cluster hire initiative. (UW–Madison’s Cluster Hiring Initiative was launched in 1998 as an innovative partnership between the university, state, and the Wisconsin Alumni Research Foundation.) The cluster created three positions to leverage and improve UW–Madison’s strengths in RNA virology, DNA virus epigenetics, and atomic-level imaging. Another goal of the cluster is to expand the institute’s research portfolio into the evolving field of metastructural virology, which studies how viruses infect and modify living cells.
Following his UW bachelor’s degree with majors in biochemistry and genetics, Kirchdoerfer earned his Ph.D. in biophysics at The Scripps Research Institute in Southern California and continued there as a postdoctoral scholar before coming back to UW–Madison.
“I was thrilled to take up this position at UW–Madison to look at the structures of macromolecular virus complexes and, particularly, look at virus spikes and viral replication complexes,” Kirchdoerfer says.
Kirchdoerfer studies coronavirus. Most of these viruses are not harmful, and some cause mild flu-like symptoms. However, some, such as SARS-CoV-2 (which causes the disease COVID-19) are more dangerous — they jump between animal species and find their way into humans. His research uses structural biology methods, such as cryo-EM and X-ray crystallography, combined with more traditional biochemistry approaches, to examine the protein machines of viruses in great detail. In viewing how these viral machines are put together and how they function, he’s probing for vulnerable points in the virus where vaccines and antiviral drugs could intervene.
Kirchdoerfer, whose research has taken on increased urgency over the last year due to the COVID-19 pandemic, has been doing much of his cryo-EM data collection at the Simons Electron Microscopy Center at the New York Structural Biology Center.
“We send them samples, and it’s helpful, but not as convenient as having a center here across the driveway,” he says. “We are really looking forward to the new facility at UW. Having greater access to microscope time means greater access to data.
“I also think this is going to be a huge boon to people in my lab to be trained on how to collect EM data. This will accelerate our ability to do our research. The technology and computational advancements in recent years have put cryo-EM in a place to be a game-changer for structural biology. It’s highly attractive to new faculty to have this cutting-edge resource and very attractive to students and postdocs.”
It was definitely attractive to Grant, formerly a research specialist at the Howard Hughes Medical Institute’s Janelia Research Campus, who joined the ranks of UW–Madison faculty in early 2020.
“One of the main reasons that I came to Madison was the investment made in cryo-EM, in the equipment, but also in hiring professors with a specialty in cryo-EM,” Grant says.
Grant’s first exposure to cryo-EM was in the early 2000s as an undergraduate at Imperial College in London. He was drawn to the technology’s combination of biology and computing.
Grant contributes to cryo-EM and its broader use as primary developer of a software package called cisTEM, which is used to process cryo-EM images of macromolecular complexes and obtain high-resolution 3D reconstructions from them. The software comprises a number of tools, including movies, micrographs, and stacks of single-particle images, and this creates a complete pipeline of processing steps for obtaining high-resolution, single- particle reconstructions.
“I spend half my time developing techniques and methods to improve the quality of images and results that you get from the electron microscopes and the other half collaborating with people to solve important structures,” he says. “A large part of cryo- EM is computation — the processing of images. For every day you spend on the microscope, you probably spend weeks or more on the data processing.”
Grant is excited about the growth of cryo-EM in Madison. “A lot of people are going to be drawn here because of the facility and national hub,” he says. “I’m excited about the opportunity to interact with them, something that I hope will lead to some interesting collaborations. It will be a focal point — a great place to meet people and share ideas.”
Access to a leading cryo-EM research facility also was a draw for Lim, who arrived in Madison in August 2020. Lim came to Madison from Colorado, where he pursued postdoctoral training in biochemistry and cryo-EM in the Cech lab at the University of Colorado Boulder. There, he was using cryo-EM technology to study how mammalian telomeres are regulated and achieve genome stability. Telomeres act as protective caps at the ends of chromosomes, holding genetic information in place. Without telomeres, some of the genetic information is lost every time a cell undergoes division. This loss of genetic information at the cellular level can lead to cancer and age-related diseases.
Lim developed an interest in telomere biology during his undergraduate research work in Singapore, where he grew up. This work led him to a Ph.D. in single-molecule biophysics at the National University of Singapore and then his first trip to the United States for postdoctoral training in Colorado.
Lim, who helps teach a course at UW in single molecule biophysics, is eager to develop more courses on this topic so he can contribute to the university’s long-standing tradition of training and mentoring the next generation of scientists in his field.
“The field of telomere biology is very specialized,” he says. “But in the broader field of structural biology, cryo-EM, as a key methodology, is important, and students need to know about it. I’m excited to share the applications of cryo-EM in biology. It’s very visual — and seeing is believing.”
Extension of the Artist’s Eye
Like Lim, Wright has a passion for teaching, especially elementary school children. Simple microscopy lessons, she says, can allow students to use their eyes, magnifying glasses, and microscopes to see the same object at different scales and resolution levels. With these snapshots, Wright instills a sense of wonder and curiosity about the complexity of living things.
It’s a wonder that Wright has felt much of her life. She studied chemistry and biology as an undergraduate in Georgia, and she made science her career, but she also took just about every art class that was offered on the way. Today, her hobbies include drawing, painting, and photography. She says imaging technologies like cryo-EM help her retain her artistic eye.
“Art, like the technology that we are using in the lab, helps us to see the world in a different way,” Wright says, “and sometimes that is beautiful and life changing.”