Electron Cryomicroscopy Takes 3D Portrait of Virus

Researchers at Purdue University have used the emerging technique of single-particle electron cryomicroscopy to get images of a virus in twice the detail than had previously been possible.

The image of the bacteriophage Epsilon15 is at a resolution of 4.5 angstroms. Approximately 1 million angstroms would equal the diameter of a human hair. In other words, they can take pictures of objects 4 ½ millionths the width of a human hair. (Hi-Res Full screen image)

This imaging technique, called cryo-EM, has the additional advantage of being able to keep the sample being observed in a state similar to its natural environment, according the study[1], published in the journal Nature. Comparable imaging techniques, such as X-ray crystallography, call for the sample to be manipulated and rotated[2].

Near Atomic Level Resolution

“This is one of the first projects to refine the technique to the point of near atomic-level resolution,” said Wen Jiang, lead researcher. “This breaks a threshold and allows us to now see a whole new level of detail in the structure. This is the highest resolution ever achieved for a living organism of this size.” Details of the structure of a virus provide valuable information for development of disease treatments.

“If we understand the system – how the virus particles assemble and how they infect a host cell – it will greatly improve our ability to design a treatment,” Jiang said. “Structural biologists perform the basic science and provide information to help those working on the clinical aspects.”

The method represents a new approach for modeling the structure of proteins in other macromolecular assemblies, such as DNA, at near-native states, Jiang said:

“The sample is purified in a solution that is very similar to the environment that would be found in a host cell. It is as if the virus is frozen in glass and it is alive and infectious while we examine it. This demonstration shows that cryo-EM is doable and is a major step in reaching the full potential of this technique,” he said. “The goal is to have it reach a 3 to 4 angstrom resolution, which would allow us to clearly see the amino acids that make up a protein.”

Electron Cryomicroscopy

In electron microscopy, a beam of electrons takes the place of the light beam used in a conventional microscope. The use of electrons instead of light allows the microscope to “see” in much greater detail.

Cryo-EM cools specimens to temperatures well below the freezing point of water. This decreases damage from the electron beam and allows the specimens to be examined for a longer period of time. Longer exposure time allows for sharper, more detailed images.

Researchers using cryo-EM had obtained images at a resolution of 6-9 angstroms but could not differentiate between smaller elements of the structure spaced only 4.5 angstroms apart.

“There are different elements that make up the protein building blocks of the virus,” Jiang said. “It is like examining a striped blanket. From a distance, the stripes blur together and the blanket appears to be one solid color. As you get closer you can see the different stripes, and if you use a magnifying glass you can see the strands of string that make up the material. The resolution needs to be smaller than the distance between the strands of thread in order to see two separate strands.

By being able to zoom in, researchers were able to see components that blurred together at the earlier achieved resolution.”

Computer programs are used to extract the signal from the microscope and to combine thousands of two-dimensional images into an accurate three-dimensional image that maps the structure of the virus. This requires use of a large data set and could not have been done without the resources of Purdue’s Office of Information Technology, or ItaP, Jiang said.

Jiang used Purdue’s Condor program – which links computers including desktop machines and large, powerful research computers – to create the largest distributed computing network at a university.

“ITaP provided us with computational power at the supercomputer scale that was necessary for this work,” he said. “Purdue’s Condor program allowed us to take advantage of the power of 7,000 computers. This was a critical element to our success.”

Jiang plans to continue to refine every step of the process to improve the capabilities of the technique and to examine more medically relevant virus species.

References

1. Wen Jiang, Matthew L. Baker, Joanita Jakana, Peter R. Weigele, Jonathan King and Wah Chiu Backbone Structure of the Infectious Epsilon15
Virus Capsid Revealed by Electron Cryomicroscopy Nature 451, 1130-1134 (28 February 2008) | doi:10.1038/nature06665

2. Ravelli RB, Garman EF (2006). “Radiation damage in macromolecular cryocrystallography”. Curr. Opin. Struct. Biol. 16 (5): 624-9. PMID 16938450.