Just as breaking the sound barrier once obsessed scientists (and airplane pilots) during the previous century, pushing past the resolution barrier enchants scientists of today.  Now, using electron cryo-microscopy (or cryo-EM), scientists at UC San Francisco (UCSF) have revealed the structure of the protein TRPV1 (pronounced "trip-vee-one"), at an awe-inspiring resolution of 3.4 angstroms. (By comparison, a single sheet of paper is roughly one million angstroms thick.) TRPV1 is known to play a significant role in our perceptions of both pain and heat.

"The impact will be broad," Yifan Cheng, Ph.D., associate professor of biochemistry and biophysics, UCSF, and co-senior author, stated in a press release. "In the past, people never believed that you would be able to use this method to get this kind of resolution — it was thought to be impossible. This opens up a lot of opportunity." The team of scientists suggest their new research, which appears in Nature, may inspire new insights among those attempting to design new drug for treating pain.

Small Scale View

The science behind cryo-EM is as fascinating to a structural biologist as the molecules viewed. To view the structure of a molecule, researchers create a sample by placing many copies of a protein, or single particles, in an aqueous solution, which is then plunged into liquid ethane. This bath cools the solution at the rate of 100,000 degrees Celsius per second and leaves particles suspended in a protective glassy ice (minus 172 degrees Celsius) at myriad angles. Next, researchers use an electron microscope to image the sample. Finally, they run their two-dimensional images of the particles through computers, which combine information from the many views to calculate the object's three-dimensional structure.

To achieve an unprecedented level of clarity as in this case, members of the research team designed a new camera to directly capture electrons rather than first converting them to light as previous cameras did.  Exploiting the new camera's 400 frames-per-second speed, Xueming Li, PhD, next devised a motion-correction algorithm, which improved the resolution even more. These hardware and software innovations resulted in significant improvement in resolution. "The picture is like a movie, and you can compensate for minute movements of the sample," Cheng said. "Now we can record every single image of the sample at 3-angstrom resolution."  Which is how the team came to view and map TRPV1.  

The Structure of TRPV1

Proteins are large, complex molecules that carry out many crucial roles and functions within living organisms. While proteins perform most of the intricate work within cells, they are also required for the structure, function, and regulation of the body’s tissues and organs. Proteins are made of hundreds or even thousands of smaller units, amino acids, which attach to one another in long chains. Twenty unique amino acids exist; it is the combination and sequence of these different amino acids that determines each individual protein’s three-dimensional structure as well as its function.

The protein TRPV1 is found in abundance in sensory nerve cells, where it forms an ion channel. This means it creates a pore in the cell membrane through which ions, such as calcium may pass, altering the cell's propensity to generate action potentials and pass on signals to other neurons. Unlike other ion channels, though, TRPV1 responds to very specific stimuli. For example, TRPV1 changes its shape to open its channel in the presence of capsaicin, the pungent compound found within fiery chili peppers, while responding much the same to temperatures high enough to elicit pain.

First identified in 1997 by Professor David Julius, senior author of the paper, TRPV1 has intrigued scientists who could prove that a range of pain-inducing toxins derived from sources as diverse as spider toxins and plants but will also activate TRPV1 while eluding them. Now, the new cryo-EM visualizations support a "two-gate" model of TRPV1 activation, which shows how different sections of the channel change shape in response to different chemical agents. Previously, Cheng noted, "the best resolution for structures of TRPV1 and similar proteins was about 15 to 20 angstroms, and many of the structures derived from the low-resolution data lacked sufficient detail to be mechanistically informative." The new data, then, could be invaluable to drug designers who hope to modulate the pain response by controlling TRPV1 gating.  


Source: Julius D, Cao E, Cheng Y, Liao M. Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature. 2013.