Microscopy at Yale has just received a major upgrade. Structural biologists at the School of Medicine and scientists from across the university have begun obtaining three-dimensional images at near-atomic resolutions from what Jorge E. Galán, Ph.D., D.V.M., chair and Lucille P. Markey Professor of Microbial Pathogenesis and professor of cell biology, calls “the mother of all microscopes.” The Titan Krios cryoelectron microscope arrived at West Campus in January and was dedicated in June. Cryo-electron microscopy (cryo-EM) is high-resolution electron microscopy of cryogenically cooled specimens. The specimens are unstained, and rapidly frozen so they are embedded in vitreous ice.
As multimillion-dollar investments in infrastructure go, bringing cryo-EM to Yale was a rapid process, according to Scott A. Strobel, Ph.D., Henry Ford II Professor of Molecular Biophysics and Biochemistry and professor of chemistry, deputy provost for teaching and learning, and vice president for West Campus planning and program development. “The West Campus had a space that was ideally suited for this instrument in terms of height, and in terms of being on bedrock,” Strobel says. While preparing that space for such a delicate installation was still a huge task, its physical configuration allowed the work to move more quickly than would have been possible elsewhere. Says Strobel, “From the time we decided to do it to the time it was in place was less than a year.”
The new device allows investigators to see structures in ways they previously could not. Its resolution rivals that of X-ray crystallography, but where crystallography requires looking at specimens in isolation, severed from biological systems of which they are a part, cryo-EM permits examination of samples in ways that better illustrate their function. Previously, researchers could only estimate the structure and function of many systems they study. Now, for the first time, they actually are seeing them.
Images with 3-angstrom (3Å) resolution are now readily available to investigators such as Frederick J. Sigworth, Ph.D., professor of cellular and molecular physiology and of molecular biophysics and biochemistry. “The difference between 5Å or 8Å [the best resolutions attainable with Yale’s prior generation of equipment] and 3Å is huge,” says Sigworth, who hopes his work on ion channel function within cells can form the basis for therapies for muscular diseases. “It’s the difference between being able to pretty well place where the atoms are in a protein versus just saying, ‘Well, roughly we’ve got this kind of shape and this kind of structure.’ If you can place all the atoms, then you can begin to think, ‘OK, how is a drug going to bind to this, or how does a hormone interact with this binding pocket to activate this receptor?’ ”
Yong Xiong, Ph.D., associate professor of molecular biophysics and biochemistry, says what investigators in Yale laboratories can see is “a revolution for us” and “will change how we do things.” Xiong’s primary work is deciphering the intricate dance between antiviral proteins in the immune system and viruses such as HIV. “We want to see how the virus infects the host, how the host tries to suppress the infection, and how the virus then develops another mechanism to escape the host’s suppression.”
He now will take advantage of the new microscope’s ability to perform cryo-electron tomography (cryo-ET), a method still in its relative infancy whose capabilities include creating high-resolution 3-D images from an unprecedented array of angles. Xiong predicts that seeing clear images of specimens in their native environment, within cells, will be a major advance. He says the standard method prior to cryo-ET has been, “We take [the specimen] out, apply an input, look at the output, and guess what it is doing. If we can use cryo-electron tomography to directly see it, that power is unprecedented.” Xiong says cryo-ET may be the sort of advance that comes along only once in a few decades.
In the lab of Charles V. Sindelar, Ph.D., assistant professor of molecular biophysics and biochemistry, one object of particular interest is the flagellum—a propeller-like molecular machine that transports pathogens through human tissue, causing diseases that include syphilis and Lyme disease. If flagella can be disabled, Sindelar explains, pathogens cannot move, so they cannot penetrate the body. “Flagella images in the past were uninterpretable,” Sindelar says. “We couldn’t translate them into a 3-D shape because they lie down in a certain way inside the microscope.” Those days, he says, are over, thanks to cryo-ET. “What we have been doing is basically tilting the stage [of the new instrument] back and forth and getting beautiful images like nothing we’ve ever seen. We could never have taken that to atomic resolution with anything except the device we have here.”
Galán proclaims that cryo-ET “is truly, truly the future.” His own work focuses on molecular machines that directly inject bacterial proteins into mammalian cells. He hopes to find ways by which cells can thwart that interaction—an approach that could be superior to attacking the bacteria, which increasingly resist antibiotics. Galán says tomography may be the key to success.
It also is key to bringing even more of the world’s top scientists to Yale. In September, Jun Liu, Ph.D., a renowned expert in tomography, will come to the School of Medicine faculty from the University of Texas Medical School at Houston, joining Galán’s Department of Microbial Pathogenesis. Says Galán of Liu, who has collaborated with Yale scientists in the past, “We’re bringing in someone who will be able to take us into the big leagues in cryo-electron tomography.”
Recruitment efforts by other departments are also well underway, spurred by the arrival of the cryo-EM. Strobel says, “We are going to be able to bring new faculty to Yale as a result of this instrument.”
As it draws top talent to Yale, the new device may also help democratize structural biology research. So intricate is the process of preparing purified samples for crystallography that, Sigworth recalls, “before the mid-1990s anyone who solved a membrane protein structure by X-ray crystallography got a Nobel Prize. It was that hard.” Preparing samples for comparable analysis by cryo-EM is far simpler. “With an automated instrument like this, solving an atomic structure is becoming so easy it can be part of a grad student’s thesis,” Sigworth says. “In fact, it could be a side project.”
“What technology is allowing us to do now is completely breathtaking,” adds Galán. “We can see microbes in action. We can see them in excruciating detail as allowed by instruments like the Titan Krios. And I think that is in essence a fantastic strength of Yale as a university, and for the medical school as well.”