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Blowing ‘Bubblegrams’ to Determine Protein Structure

Sarah C. P. Williams

When proteins are encased in DNA, as is the case for many virus capsids, it’s hard to work out the structure of those proteins. A new method solves the problem by bombarding the capsids with radiation.

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A serendipitous observation of tiny bubbles appearing under cryo-electron microscopy has led to a new way to get information about proteins that are wrapped in—or closely associated with—nucleic acids. The technique, dubbed “bubblegrams,” relies on the fact that proteins are more sensitive to radiation than nucleic acids are. And when a protein is bombarded with radiation, it releases radiation products such as free radicals.

The patterns of bubbles released by proteins during cryo-EM gives researchers more information about proteins embedded in DNA than does cryo-EM alone. Source: NIH

“Normally, these radiation products diffuse fairly freely,” explained Alasdair Steven, a structural biologist at the National Institutes of Health who led the development of the technique. “But when there’s DNA surrounding the protein, the concentration builds up until the radiation products come out of the solution as bubbles.”

The technique’s development began when Steven’s collaborator Lindsay Black at the University of Maryland School of Medicine was studying фKZ, a virus—or bacteriophage—that infects the bacteria Pseudomonas aeruginosa. He wanted to determine the structure of the bacteriophage but kept hitting a wall because the proteins that make it up are tightly packed inside dense DNA.

When фKZ is frozen in ice and imaged with the low levels of radiation typically used in cryo-electron microscopy, only the structure of the outer DNA can be seen. And when the researchers increase radiation levels, the protein that they want to visualize is quickly destroyed. But looking at the images of the proteins as they were being damaged by high radiation, Black and her lab members noticed unexpected columns of bubbles. So, Black approached Steven, who had previously studied radiation damage of proteins.

“At first they thought it was some contaminant or a chemically different substance,” said Steven. “But because this protein is embedded in DNA, I figured that probably what’s happening is that the protein’s radiation products can’t diffuse away like they usually do, so they escape periodically in bubbles.”

The patterns of bubbles—or bubblegrams—held valuable information about the proteins. Since the bubbles were released at spots where proteins, and not nucleic acids, were present, they could be use to map a protein’s arrangement inside the DNA shell. In a paper published in the January 13, 2012, issue of Science, Steven and Black used a series of bubblegrams to show, for the first time, how proteins are oriented inside фKZ’s nucleic acid capsid (1). Next, the team plans to study how this orientation or arrangement changes when фKZ is actively infecting a bacterium.

Further research on what causes the bubbling could lead to an ability to get even more detailed information about DNA-encased or DNA-bound proteins. “What we haven’t done yet is to reconstruct the bubbles. They all follow this cylindrical structure, but the specific bubbles are different from particle to particle,” said Steven. The differences could stem from specific structural elements of the proteins.

In addition, the method may be useful for gaining structural information not only about capsid-contained viruses but also the structure of chromatin—the combination of DNA and proteins in the nucleus of a cell. “There are a lot of possibilities in terms of using this to distinguish between protein and nucleic acid in any system,” Steven said.


  1. Wu, W. Thomas, J. Cheng, N. Black, L. Steven, A. 2012. Bubblegrams Reveal the Inner Body of Bacteriophage фKZ. Science 13 January 2012: 182.