Merry Microscopy and a Happy New Technique!

Color EM WreathSeasons Greetings! What looks like a humble wreath actually represents an awe-inspiring gift to biomedical research: a new imaging technique that adds a dash of color to the formerly black-and-white world of electron microscopy (EM). Here the technique is used to visualize the uptake of cell-penetrating peptides (red) by the fluid-filled vesicles (green) of the endosome (gray), a cellular compartment involved in molecular transport. Without the use of color to draw sharp contrasts between the various structures, such details would not be readily visible.

This innovative technique has its origins in a wonderful holiday story. In December 2003, Roger Tsien, a world-renowned researcher at the University of California, San Diego (UCSD), decided to give himself a special present. With the lab phones still and email traffic slow for the holidays, Tsien decided to take advantage of the peace and quiet to spend two weeks alone at the research bench, pursuing an intriguing, yet seemingly wacky, idea. He wanted to find a way to deposit ions of a rare earth metal, called lanthanum, directly into cells as the vital first step in creating a new imaging technique designed to infuse EM with some much-needed color. After the holidays, when the lab returned to its usual hustle and bustle, Tsien handed off his project to Stephen Adams, a research scientist in his lab, thereby setting in motion a nearly 13-year quest to perfect the colorful new mode of EM.

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Snapshots of Life: A Flare for the Dramatic

lipid-covered water drop

Credit: Valentin Romanov, University of Utah, Salt Lake City

Oil and water may not mix, but under the right conditions—like those in the photo above—it can sure produce some interesting science that resembles art. You’re looking at a water droplet suspended in an emulsion of olive oil (black and purple) and lipids, molecules that serve as the building blocks of cell membranes. Each lipid has been tagged with a red fluorescent marker, and what look like red and yellow flames are the markers reacting to a beam of UV light. Their glow shows the lipids sticking to the surface of the water droplet, which will soon engulf the droplet to form a single lipid bilayer, which can later be transformed into a lipid bilayer that closely resembles a cell membrane. Scientists use these bubbles, called liposomes, as artificial cells for a variety of research purposes.

In this case, the purpose is structural biology studies. Valentin Romanov, the graduate student at the University of Utah, Salt Lake City, who snapped the image, creates liposomes to study proteins that help cells multiply. By encapsulating and letting the proteins interact with lipids in the artificial cell membrane, Romanov and his colleagues in the NIH-supported labs of Bruce Gale at the University of Utah and Adam Frost at the University of California, San Francisco, can freeze and capture their changing 3D structures at various points in the cell division process with high-resolution imaging techniques. These snapshots will help the researchers to understand in finer detail how the proteins work and perhaps to design drugs to manipulate their functions.

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Creative Minds: Breaking Size Barriers in Cryo-Electron Microscopy

Dmitry Lyumkis

Dmitry Lyumkis

When Dmitry Lyumkis headed off to graduate school at The Scripps Research Institute, La Jolla, CA, he had thoughts of becoming a synthetic chemist. But he soon found his calling in a nearby lab that imaged proteins using a technique known as single-particle cryo-electron microscopy (EM). Lyumkis was amazed that the team could take a purified protein, flash-freeze it in liquid nitrogen, and then fire electrons at the protein, capturing the resulting image with a special camera. Also amazing was the sophisticated computer software that analyzed the raw 2D camera images, merging the data and reconstructing it into 3D representations of the protein.

The work was profoundly complex, but Lyumkis thrives on solving extremely difficult puzzles. He joined the Scripps lab to become a structural biologist and a few years later used single-particle cryo-EM to help determine the atomic structure of a key protein on the surface of the human immunodeficiency virus (HIV), the cause of AIDS. The protein had been considered one of the greatest challenges in structural biology and a critical target in developing an AIDS vaccine [1].

Now, Lyumkis has plans to take single-particle cryo-EM to a whole new level—literally. He wants to develop new methods that allow it to model the atomic structures of much smaller proteins. Right now, single-particle cryo-EM has worked with proteins as small as roughly 150 kilodaltons, a measure of a protein’s molecular weight (the approximate average mass of a protein is 53 kDa). Lyumkis plans to drop that number well below 100 kDa, noting that if his new methods work as he hopes, there should be very little, if any, lower size limit to get the technique to work. He envisions generating within a matter of days or weeks the precise structure of an average-sized protein involved in a disease, and then potentially handing it off as an atomic model for drug developers to target for more effective treatment.

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Snapshots of Life: Imperfect but Beautiful Intruder

RSV Particle

Credit: Boon Chong Goh, Beckman Institute, University of Illinois at Urbana-Champaign

The striking image you see above is an example of what can happen when scientists combine something old with something new. In this case, a researcher took the Rous sarcoma virus (RSV)—a virus that’s been studied for more than century because of its ability to cause cancer in chickens and the insights it provided on human oncogenes [1, 2]—and used modern computational tools to generate a model of its atomic structure.

Here you see an immature RSV particle that’s just budded from an infected chicken cell and entered the avian bloodstream. A lattice of proteins (red) held together by short peptides (green) cover the outer shell of the immature virus, shielding other proteins (blue) that make up an inner shell.

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LabTV: Curious about Computer Modeling of Proteins

Josh Carter

In many ways, Josh Carter is a typical college student, with a hectic schedule packed with classes and social activities. But when he enters a structural biology lab at Montana State University in Bozeman, Carter encounters an even faster paced world in which molecular interactions can be measured in femtoseconds—that is, 1 millionth of 1 billionth of 1 second.

Working under the expert eye of principal investigator Blake Wiedenheft, Carter is applying his computational skills to X-ray crystallography data to model the structures of various proteins, as well as to chart their evolution over time and map their highly dynamic interactions with other proteins and molecules. This basic science work is part of this NIH-funded lab’s larger mission to understand how bacteria defend themselves from the viruses that try to infect them. It’s a fascinating area of science with a wide range of potential applications, from treating diseases that arise from imbalances in the microbiome (the communities of microbes that live in and on our bodies) to developing new methods for gene editing and programmable control of gene expression.

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