A dedicated researcher and a big magine team-up to depict tiny enzymes.

This technology, called x-ray crystallography, has been a mainstay of science for the past century. As it has advanced, it’s become essential to scientists trying to determine the chemical structure of many biological molecules.

MU is part of the Molecular Biology Consortium, a group of 12 research universities that share an automated laboratory at the Berkeley synchrotron where they do their x-ray crystallography studies. MU has its own x-ray crystallography facilities, but the strength of their x-rays are just a fraction of what the Berkeley synchrotron can produce. An experiment that would take three minutes at Berkeley would take three days at MU. “It’s bigger, better and faster in California,” Tanner says.

Tanner is using liquid nitrogen at minus 321 degrees Fahrenheit to freeze the crystals. They have to be frozen cryogenically this way to withstand the synchrotron’s intense radiation. As he takes the containers of enzyme crystals out of a small insulated tank of boiling nitrogen, some of the liquid splashes onto the floor, where it steams and quickly evaporates.

The enzymes Tanner investigates are proteins that serve as catalysts to make chemical reactions go faster. “Literally tens of thousands of enzymes are needed to keep us alive,” he says.

Tanner’s lab studies enzymes that participate in cell metabolism, and in particular, a class of enzymes that are especially abundant in cancer stem cells. Cancer stem cells are normal stem cells’ evil twin. Just as normal stem cells are able to differentiate into a variety of cells, cancer stem cells create new cancer cells. Most cancer chemotherapy targets non-stem cells. The medications may shrink a tumor significantly, but still leave a residue of the stem cells.

“It may look really good at first, but these stem cells can differentiate into other cell types and the cancer returns,” Tanner says.

The goal of Tanner’s stem cell project, in collaboration with MU organic chemist Kent Gates, is to try to make small-molecule compounds that bind to these enzymes, jamming their function to keep them from working in the stem cells. Ultimately, that could lead to chemotherapy targeted to these cancer cells.

As the minutes tick by, Tanner uses tongs to methodically insert small tubes, each holding a crystal, into holes in five-inch-diameter cylindrical metal cassettes that look like hockey pucks. These “pucks” eventually will be loaded into the automated instruments at the Berkeley synchrotron that will give Tanner the raw data he needs to unlock the enzymes’ structure.

“The credo in my lab is ‘if you can see what an enzyme looks like you can start to understand how it works,’” Tanner says. He compares it to looking at a car engine. “We pop the hood on the enzyme to see how it works.”

Earlier in the afternoon, Tanner put an image of one of the enzymes he is studying, aldehyde dehydrogenase 7, on the computer screen in his office.

It’s a blue lattice of various lumpy shapes of different sizes joined together. The size and shape of the lumps show the levels of electron density in the different parts of the molecule. Tanner can use his mouse and keyboard to move around the protein and rotate it to view it from different angles, a cosmos of 2,000 amino acids comprising 20,000 atoms.

Put on a pair of 3-D glasses and the image leaps out from the computer screen, making the complexity of its shape all the more apparent. “Protein structure is an extraordinarily information-dense network,” he says.

Scientists can make sense of these images because all amino acids, the building blocks of proteins, have characteristic shapes. In the early 1990s, scientists like Tanner had to match the shapes, with their often subtle differences, to tryptophan, phenylalanine, lysine and other amino acids.

“I’d go hunting around for a starting point,” he says. Often, he’d start looking for tryptophan because it is large enough to stick out from the other amino acids. “It seems impossible, but it’s really just a shape recognition problem.” Now computers help with that work.

Once the amino acids are identified, their chemical structures can be superimposed onto the computer image, creating a kind of skeleton within the enzyme. The amino acids have to link together in a way that makes sense chemically. It is a bit like breaking a code while solving a jig-saw puzzle.

“In the end, the message has to make sense. If it doesn’t, you probably made a mistake,” Tanner says. “It’s a really satisfying moment when you put the last piece in and it fits.”

As Tanner loads the crystals into the “pucks,” he keeps a careful inventory of the crystals. The pucks are color-coded, blue, gold, red and other hues, to make them easier to track. This will all be essential to manage the massive amounts of data he’ll get back once the pucks are loaded into the robot at the Berkeley lab.

The Lawrence Berkeley Laboratory’s Advanced Light Source synchrotron is housed in a 1940s-era building. Construction on the synchrotron started in the late 1980s, and was completed in 1993. It is one of only five such synchrotron in the nation.

Its ring of superconducting magnets keeps a hair-thin beam of electrons accelerating around a circle, so fast that they produce x-rays brighter than the sun. Beam lines of x-rays shoot off the ring where they are picked up at about 20 small laboratories that circle the ring. The work leading to Berkeley starts back in Tanner’s lab, where researchers grow crystals of the proteins they want to study, a time-consuming, laborious process. “It’s as much art as it is science,” Tanner says.

The refrigerator in his lab holds stacks of plastic trays holding crystals bathed in a protein solution waiting to be frozen and shipped to Berkeley. In the synchrotron laboratory, each frozen crystal is slowly rotated by a goniometer as it is zapped by x-rays that diffract into patterns of millions of dots. Encoded in these dots are the densities of the electrons in different parts of the protein molecule. These the computer translates into images. It’s analogous to the way a CT scanner circles around the body to create three-dimensional digital images.

Eleven other universities, including Washington University, the University of Iowa, the University of Minnesota, Oregon Health Sciences University, Loma Linda University and the University of Texas at Houston, had agreed to build the lab at the Berkeley synchrotron before MU came on board.

MU agreed to participate on the condition that the lab would ultimately be automated. This was Tanner’s idea. He had been reading about how astronomers were using large telescopes remotely. “I thought ‘this is exactly what we should be doing.’”

Gerald Hazelbauer, Curators’ Professor and chair of the biochemistry department, was spearheading MU’s effort to join the beamline consortium. Tanner advised him that MU’s $500,000 share of the lab’s cost should go toward robotics. Hazelbauer made it happen by arguing that the investment put MU on the cutting edge of biomedical research, Tanner recalls. The beamline, Hazelbauer said, was also key to attracting new faculty in structural biology.

In the beginning, before the robot was installed, Tanner and other scientists had to travel to Berkeley to conduct their experiments. It was fun to visit, he says, but it was an expensive and time-consuming way to do science. “I knew I’d been going a lot when I’d see the same stranger on the same flight,” he says.

About seven years ago, the universities installed the robot arm that can be remotely controlled to pick up crystals and place them in the beam. The lab’s one full-time staffer is its manager, Jay Nix.

“It’s really just Jay, the robot and, occasionally, a part-time Berkeley undergrad helping,” Tanner says. “That robot is the key to making this work. I can run data from home. I’ve sat at a bar and collected data while drinking beer. That’s the ultimate way to do science.”

Tanner hadn’t always planned on a career as a biochemist.

He grew up in a working class family in St. Louis. His father drove a milk truck. His mother was a librarian and a voracious reader. Tanner loved numbers and excelled in problem solving. When he left home for MU, he expected to go into engineering. That didn’t last long. “I was more interested in things you couldn’t see, like molecules, and in solving mysteries,” he says.

So he moved on to physical chemistry. As a doctoral student at Brown University in Providence, R.I., he studied theoretical chemistry and quantum mechanics. “It was exciting when I was a grad student. Then you pause and consider, ‘What is the frontier?’ And I decided the vanguard of the field was not there,” he says. “The field I got my degree in was already very mature when I went in. The frontier was biology and biochemistry.”

From Brown, Tanner went southwest to the University of Houston for two post-doctoral appointments that would take him in that direction.

The first was in computational biology, a field that uses massive amounts of data to create mathematical models that can evaluate how species evolve over time, for example, or analyze the molecules responsible for causing cancers.

Tanner’s second post-doc was in x-ray crystallography. Until that time, he had been doing “virtual experiments” on computers. But a member of the faculty was starting a new laboratory and “he took a chance on a guy who had never done an actual experiment.”

He spent nine years at Houston. It is where he met his wife. “We met the old fashioned way, at a bar,” he says. They now have a daughter, 14, and a son, 12.

Tanner came to MU in 1997, a time when the University was recruiting faculty to build a program in structural biology. It was a nostalgic homecoming. The first MU class he taught was in the same auditorium where he took his first chemistry class. The lab he uses today started off as a classroom lab where he took organic chemistry.

As he talks, Tanner puts pucks loaded with crystals bound for Berkeley into a metal cylinder and then submerges it back into liquid nitrogen. He says he didn’t initially set out to study these enzymes because of their importance to cancer research. His interest was in enzymes involved in metabolic pathways. Serendipitously, some of these enzymes turned out to have potential medical applications.

“I think it’s a good example of how science really works,” Tanner says. “You study something because it’s interesting. You’re creating knowledge, and it turns out it’s important for something else.”

HIV is a great example of this approach, he says. Before AIDS, researchers already were studying retroviruses. That speeded development of diagnostic tests for HIV, as well as anti-retroviral drugs to fight the disease. “Imagine if all that research hadn’t been done. You’d be starting at ground zero.”

“It makes it hard to take a top-down approach to science,” Tanner says. “But I fear that’s what’s happening in the U.S.,” as science funding is directed more and more toward research oriented towards an immediate payout.

Besides a passion for baseball — he’s a big Cardinals fan — and an interest in vintage hi-fi equipment, Tanner’s attentions are focused on his home and his laboratory. “I pretty much work all the time,” he says.

It’s a routine of purifying proteins, growing them into crystals, freezing them, shipping them to Berkeley, then decoding the results. All the work, he says, comes down to what first drew him to structural biology and the high-tech tools of x-ray crystallography in the first place: the chance to solve mysteries, to see the unseen, to explore the structure of each newly revealed protein.

“When you’re the first person in history to see that,” Tanner says, “that’s exciting.”

The frozen crystals are all packed into a foam-lined plastic box. Labels are attached. Once the box arrives in Berkeley, it will take about 20 hours to collect data on all 60 of the crystals. But here in Columbia, it’s a quarter of four. The delivery truck has already gone, so this work day just got a little bit longer. Tanner will be driving his crystals to the Fed Ex office on his way home.

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