'Blobology' no more

Cryo-EM microscopy opens up biology at atomic scales
Dr. Melody Campbell prepares a sample under the Cryo-EM microscope
Dr. Melody Campbell is scientific director of the new cryo-EM microscope system at Fred Hutchinson Cancer Research Center. Above, she prepares a sample for imaging. Photo by Robert Hood / Fred Hutch News Service

Switchblade. Headpiece. Propeller. Ankle. Calf. Thigh.

Such are the pet names for the body parts of integrins, sticky and flexible proteins that blanket the surfaces of white blood cells, which are key components of the human immune system.

Dr. Melody Campbell at Fred Hutchinson Cancer Research Center is an expert on integrins and their various moving parts. She observes these tiny structures all the time, their computer-generated images streamed to her screens via cryogenic electron microscopy.

Using specimens flash-frozen to -292°F, cryo-EM may be the coolest topic in biology today. Following a string of advances that began in the 1980s, cryo-EM has improved our ability to see biological molecules so clearly that staid academic journals now routinely describe it as revolutionary. In 2017, three early pioneers of this technology shared the Nobel Prize in chemistry.

As scientific director of the newly installed $4 million cryo-EM system at Fred Hutch, Campbell can call up images of integrins at near-atomic resolution — clear enough to discern in computer-enhanced images the molecular backbones that define the shape and reveal the functions of these important proteins. With 3D images showing the structure of these proteins, scientists can visualize their moving parts, how they interact and even connect with other proteins, how they carry out helpful work, or how their harmful activities might be blocked.

Cancer researchers are especially interested in integrins, as they may play a role in suppressing or promoting tumor development. Campbell uses cryo-EM imagery to understand the mechanics of integrins. Their primary jobs are to help normal cells bind to the structural features of their immediate environment and to pass chemical signals back and forth, within such cells and throughout their surroundings.

Drs. Melody Campbell (left) and Barry Stoddard (right) use advanced technologies to solve the structures of proteins.
Drs. Melody Campbell (above) and Barry Stoddard (below) use advanced technologies to solve the structures of proteins.

Photos by Robert Hood / Fred Hutch News Service

Researchers are using cryo-EM to explore the details of how integrins are linked to cancer, and there is much we still do not fully understand.

“We know that up-regulation of integrins is found in certain tumor cells, and that by inhibiting certain integrins tumor growth can be slowed,” Campbell said. “But we don’t know the precise details: Whether it is direct, or through regulation by other proteins, is unknown.”

The colorful shorthand for the various limbs of integrins grew out of decades of work — using earlier versions of electron microscopy and other imaging technologies — trying to understand how these strange, multi-jointed structures operate.

Campbell spent the past decade at the Scripps Research Institute in La Jolla and the University of California at San Francisco before joining the Hutch last fall. As such, her career path brought her to cryo-EM just as the full potential of this technology was being realized. Her predoctoral training began when the imagery once derided as “blobology” was coming into focus to reveal detailed, complex protein structures. Today they can show, in some cases, the positions of individual atoms within protein molecules.

In fact, the rise of cryo-EM came in chapters that stretch back to the 1970s, as various inventions and innovations subtly improved the technology and things it became capable of achieving.

A massive database of protein structures

Structural biologist Dr. Barry Stoddard, who has been resolving the shapes of proteins since he joined the Basic Sciences Division of Fred Hutch in 1992, said there has been a “sea change” in the amount and complexity of information generated in the field and in how that data can be analyzed.

“When I was a graduate student in 1985, I was able to find and photocopy almost every important paper describing a protein structure in a single, long afternoon in the MIT library. Today, there are almost 200,000 proteins in the protein structural database,” he said. Until recently, nearly all those structures were solved by a process known as X-ray crystallography.

Much of his work involves that painstaking process of isolating and forming purified crystals of proteins of interest, then bombarding those crystals with brief but powerful bursts of X-rays. The actual shape and structure of those proteins can be inferred by sophisticated analysis of the patterns of light scattered by those crystals.

It is related to the process used by Rosalind Franklin in 1952, when she produced an X-ray photograph of crystalized DNA. It revealed the famed “double helix” structure of those molecules, subsequently described by her colleagues Drs. Francis Crick, James Watson and Maurice Wilkins — for which they were awarded the Nobel Prize in physiology or medicine ten years later.

Although Stoddard built a career using X-ray crystallography — and continues to use it to solve the structure of small proteins — he has become a convert to the use of cryo-EM for the speed and ease with which it can reveal the structure and functions of larger proteins and those that are too big, flexible or complex to be crystallized.

“Cryo-EM is revolutionizing structural biology,” Stoddard said.

That technology now defines the leading edge of microscopy, a field that began in a different world. Using a primitive microscope of his own design, Dutch lens maker Antonie van Leeuwenhoek began in the 1670s to discover tiny life — "little animals" or "animalcules" as translated into English — in pond water samples. Famous in his day, he made important discoveries of bacteria and other microbes and is considered the father of microbiology.

Left: Portrait of Antonie van Leeuwenhoek. 1686. Center: Drawings of microbes from his book, "Arcana Naturae Detecta." Right: A copy of a van Leeuwenhoek simple microscope
Left: Portrait of Antonie van Leeuwenhoek. 1686. Top: Drawings of microbes from his book, "Arcana Naturae Detecta." Bottom: A copy of a van Leeuwenhoek simple microscope.

Images courtesy Wellcome Trust. Credits as follows: (portrait) J. Verkolje, 1686. (book) Antonie van Leeuwenhoek. (microscope) Leyden, 1901-1930.

Fast-forward to the 1930s, when scientists developed electron microscopes, which use a beam of electrons rather than light to see objects as small as atoms in the structures of materials. While electron microscopes made such extraordinary resolution possible for physicists, at first they were not of much use in biology. Strong electron beams will quickly destroy the fragile proteins found in living cells. They were great for crisp and scary close-ups of dead spiders, but useless for probing the delicate intricacies of biomolecules.

SEM image of a spider
A scanning electron microscope image of a spider. Cryo-EM is far more powerful, able to produce images of proteins at atomic scale. Getty Images stock photo

Microbiologists nevertheless persisted in finding ways to observe samples of life in modified electron microscopes, and then in the 1980s began a series of stepwise improvements that gradually honed the capabilities of this technique, leading to important advances that in 2017 won the Nobel Prize in chemistry for Drs. Jacques Dubochet, Joachim Frank and Richard Henderson.

Key developments include flash-freezing purified samples of protein structures in a bath of liquid ethane. The shock of this plunge into an extremely cold bath locked biomolecules in place without forming ice crystals that otherwise can crush or distort them. The results in the mid-1980s were blurry but distinguishable images of viruses and proteins on the surfaces of bacteria.

A 1987 image of a Sindbis virus
A state-of-the-art cryo-EM image of the mosquito-borne sindbis virus, published in 1987. [click on image for link to journal article] Fuller, SD (1987) The T=4 envelope of sindbis virus is organized by interactions with a complementary T=3 capsid. Cell. 10.1016/0092-8674(87)90701-X.

Cryo-EM researchers also fine-tuned “stages,” the platforms that hold the frozen specimens in place in the path of the electron beams. When flash-frozen, protein specimens are often randomly oriented in the layer of ice and each micrograph shows many different angles of the same protein. Collecting thousands of micrographs with sometimes millions of particle images yields the kind of data that allows structural biologists to create 3D models of viruses and proteins. It sounds simple, but the experts have been tweaking and improving their tilt techniques for nearly 40 years, with incremental gains. Today, aided by superfast computers and sophisticated image-processing algorithms, they just keep getting better at it.

Hepatitis core particles
Images of hepatitis B virus particles created using cryo-EM and 3D image processing in 1994. [click on image for link to journal article] Crowther, RA (1994) Three-dimensional structure of hepatitis B virus core particles determined by electron cryomicroscopy. Cell. doi: 10.1016/0092-8674(94)90142-2.

Even into the 2000s, however, the structures looked more like rubber toys that the molecules of life. X-ray crystallographers were setting the standards for protein modeling of increasingly complex 3D models that resembled lattices made of noodles. With the ability to discern the location of individual atoms of a molecule, their models were hard to build but useful in understanding how proteins interacted with one another, how they folded, or how they could be blocked by a small-molecule drug or antibody.

In contrast, for their pudgy images of viruses, those pioneering cryo-EM scientists were teased as practitioners of blobology.

But the goal of cryo-EM researchers was to keep pushing the resolution limit down. The goal was to be able to see the structures of small-molecule drugs, water molecules and metallic ions that interact with proteins. In the mid-1990s, the best that could be accomplished in cryo-EM was to be able to resolve structures of about 3 nanometers, or 30 angstroms, which was already well in the range of X-ray crystallographers.

Aquareovirus cryo-EM image
This image of an aquareovirus particle was achieved using cryo-EM at 3.3 angstrom resolution in 2010. (click on image for link to journal article) Zhang, X. 3.3 A cryo-EM structure of a noneveloped virus reveals a priming mechanism for cell entry. Cell. 10.1016/j.cell.2010.03.041.

In Campbell’s view, a true revolution in the field accompanied the adoption, by 2010, of advanced cryo-EM cameras. Until this advance, the electron beams that penetrated and scattered after striking the frozen proteins had to be converted into light (photons), so that cameras could record these images. It was an inefficient process that made it harder to map fine structures within proteins. But the new cameras could directly read the electrons, without conversion to light. It created a new level of clarity and detail. That is when structural biologists all over the world realized that big changes were at hand.

Also, increasingly sophisticated computer programs could analyze tens of thousands of protein images, yielding a kind of computer consensus image or average, representing the most likely structure of a single protein particle.

By 2012, this technology led researchers, including then-graduate student Campbell, to use the improved frame-rate of this new camera to collect a series of images and to string them together into “movies” that could be further analyzed by specialized software that she and her colleagues wrote. They are yielding finely detailed models of complex, dynamic proteins like integrins, offering understanding about the form and function of their moving parts.

Integrin-mediation activation of TGF-beta w/o diffusion
Dr. Melody Campbell was lead author of a paper in Cell in 2020 that included this graphic using cryo-EM images. It shows how alpha(v)beta8, an integrin protein on the surface of a cell (bottom) binds delicately with an important immune signaling protein, TGF-beta, on another cell surface (above). The mechanics are such that it can do so without triggering release of toxic levels of TGF-beta, a potentially important finding for the use of immune modulating drugs to fight cancer. (click on image to link to journal article) Campbell, M. (2020) Cryo-EM Reveals Integrin-Mediated TGF-beta Activation without release from Latent TGF-beta. Cell. 10.1016/j.cell.2019.12.030.

The march toward higher and higher resolution approaching atomic scale was transformative, opening up new possibilities for researchers, as cryo-EM trailblazer Dubochet would explain in his 2017 Nobel Prize lecture. When “atoms become visible … blobology becomes chemistry,” he said.

“Chemistry is a powerful science,” he added. “When the arrangement of atoms can be visualized, the possibility to act on them is not very far from our reach.”

For structural biologist Stoddard, the proof was in the pudding. For more than 10 years, his laboratory had been stymied in an effort to use X-ray crystallography to solve the structure of enzymes such as DrdV, which are all-important proteins used by cells to defend against viral invaders. After switching to cryo-EM, he said the project was quickly on track.

“In less than a year, we solved the structure of DrdV, and caught this protein in the act of binding DNA,” he told colleagues in a recent lecture. 

Shen/Stoddard Cell June 2021 Coordination of phage genome degradation
Using cryo-EM, Dr. Barry Stoddard and colleagues produced these 3D images revealing the protein structure of DrdV, a DNA-cutting enzyme. (click on image to link to journal article) Shen, BW. (2021) Coordination of phage genome degradation versus host genome protection by a bifunctional restriction-modification enzyme visualized by cryo-EM. Structure. 10.1016/j.str.2021.03.012

Stoddard and his team published a paper in Structure in June describing how they not only modeled the structure of DrdV, but now understood the step-by-step process by which this active bacterial protein can degrade the DNA of invading viruses.

As a long-time X-ray crystallographer, Stoddard said he will continue to use that technology to solve problems into the future, but he also foresees a golden age for biology because cryo-EM technology is easier to use and can achieve things crystallography cannot. In a sense, these new microscopes are democratizing structural biology.

“The entire field is getting more simple and accessible to biologists of all training,” Stoddard said.

Sabin Russell is a staff writer at Fred Hutchinson Cancer Center. For two decades he covered medical science, global health and health care economics for the San Francisco Chronicle, and wrote extensively about infectious diseases, including HIV/AIDS. He was a Knight Science Journalism Fellow at MIT, and a freelance writer for the New York Times and Health Affairs. Reach him at srussell@fredhutch.org.

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