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19 October 2018

Research leading to practical benefits for society.

To market, to market, to buy a teeny tiny molecule?

Hafenstein (left) consults with assistant research professors Jennifer Gray (center) and Danielle Hickey (right) in the main control room for the Titan/Krios microscope. Credit: Nate Follmer, Penn State.
Hafenstein (left) consults with assistant research professors Jennifer Gray (center) and Danielle Hickey (right) in the main control room for the Titan/Krios microscope. Credit: Nate Follmer, Penn State.
Structural biologists aim to understand the shape, conformational changes, and interactions of microscopic molecules. While single molecules are invisible to the naked eye, they form the foundation of cellular structure, metabolism, and ultimately human health. For example, understanding molecular structure and behavior lends critical insight into how diseases, such as malarial or viral infections, can be treated or prevented.

One of the most exciting new techniques to help scientists visualize molecules and their interactions is cryo–electron microscopy, or cryoEM. Utilizing a transmission electron microscope, cryoEM requires relatively minimal sample preparation and can yield atomic-level resolution (see “Resolution Revolution”).

At this time, cryoEM is compatible with resolving large (>200kD), hydrophilic, and symmetrical proteins. While many interesting proteins and protein complexes fall within these parameters, there is a vast array of important molecules that do not. Molecules that are commonly incompatible with current cryoEM methods include small (<120kD) and hydrophobic proteins, lipids, small inorganic molecules, and carbohydrates.

CryoEM’s current limitations can pose a significant hurdle for researchers. For example, Scott Lindner, assistant professor of biochemistry and molecular biology in the Eberly College of Science, studies transmission of the malaria parasite (Plasmodium spp.) and seeks to understand the proteins necessary to the infection process. These proteins represent potentially valuable novel drug targets to prevent or halt a malarial infection. However, many of the proteins critical to malarial transmission are too small for current cryoEM techniques. These proteins are also not amenable to other assays common to structural biology, such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy.

The Lindner lab is not alone in this struggle to study small proteins. Susan Hafenstein, associate professor of biochemistry and molecular biology in the Eberly College of Science, seeks to characterize viral proteins that are far smaller than the existing limits of cryoEM.

In order to solve this problem, Hafenstein and Lindner formed a collaboration. While their initial aim was to develop a tool to resolve small proteins, what they ended up inventing is a novel, versatile, and easy-to-use platform that will enable researchers to study a wide range of molecules that are currently unsuitable for cryoEM.

At first glance, Hafenstein and Lindner’s platform seems to be no more than a simple cage. Made of self-assembling protein subunits, the cage somewhat resembles an open-sided soccer ball, and this shape is critical to the tool’s function. As a sample is prepared for imaging, the cage can roll freely through the sample medium. But once a sample is frozen, the cage is stopped mid-roll. The round shape allows the cage to be frozen in a variety of positions and ensures that the tool can be imaged from every side. This depth of imaging is necessary in order to construct a high resolution, three-dimensional (3D) representation of the cage.

Embedded in each side of the cage are five identical binding sites. Each of these sites binds a molecular “tag.” This tag effectively acts as bait, allowing the molecule of interest to be efficiently captured by the matching binding site. Five binding sites per side means up to 60 molecules can be captured per cage. This redundancy increases the chances of successful binding and increases the number of times the molecule of interest can be imaged in a single sample. Coupled with the cage’s round shape and ability to fall in a wide range of orientations, the molecule of interest can be imaged from almost every angle, allowing researchers to reconstruct its 3D shape with atomic-level resolution.

Perhaps the best part of this novel technology is how easy it is to use. To prepare a sample for imaging, all a researcher needs to do is add their tagged molecule of interest to the cage and then mix for up to two hours. Mixed samples can then progress directly to standard cryoEM processing.

Hafenstein and Lindner have created cages with binding sites that are compatible with two popular types of tags. They believe that these binding sites can be easily modi ed, allowing different types of tags to also be used. In proof-of-concept studies, the two inventors successfully imaged the cage via cryoEM, creating a full 3D reconstruction of the cage and achieving close to atomic-level (5Å) resolution. Through ongoing experiments, Hafenstein and Lindner seek to further improve this limit of resolution.

Development of this cage technology and accompanying methods was funded by a Lab Bench to Commercialization Grant. This grant, co-funded by the Eberly College of Science, College of Medicine, and Penn State Research Foundation, provides $75,000 to promote translational research, with the primary aim of moving technologies towards commercialization.

By Melissa Long

To learn more about this grant program or the described technology, please contact the College of Science Office for Innovation at innovation@science.psu.edu or 814-867-6287.