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How the Method Works
Subramaniam is helping the NCI intramural program expand its use of cryo-EM. The Center for Molecular Microscopy (https://cmm.nci.nih.gov/), which he directs, hosts both transmission and focused ion beam microscopes and works with intramural collaborators to study cells, viruses and molecules in 3D at high resolution.
The contrast is striking. The cryo-EM microscope that the Subramaniam team uses for high-resolution structural work, a Titan Krios manufactured by FEI Co., stands more than 13 feet high and weighs in excess of 2,000 pounds. It’s designed to study individual molecules and even visualize single atoms—material far too tiny to be seen without high-tech help.
The first step in generating a structure, says Subramaniam, is suspending the chosen molecules in a solution and placing them on a fine mesh screen called a “grid.” Next the grid is plunged into liquid ethane to form a thin film of ice with the proteins in it. The film gets inserted into a cassette, which then goes into a cryo-capsule device that gets inserted into the Titan. Next, the microscope takes thousands of pictures, delivered every 60 seconds or so.
Because of the way electrons can interact with biological molecules like proteins, it can be complicated to get a very clear signal in any single image. In addition, all those images are in 2D. So to get a 3D picture of the protein at atomic resolution, “we [computationally] assemble all of those pictures together,” Subramaniam explains. When there’s enough information and everything is done correctly, “out comes the structure of the protein” at atomic resolution.
Cryo-EM is still mostly uncharted territory, however.
Illustration of the structure of p97, a target for cancer therapy, trapped in an inactive state by a newly designed inhibitor. The structure is a composite of multiple states derived by cryo-EM analysis, blended to highlight the dynamic nature of this molecular machine.
Photo: Veronica Falconieri
“We need to look at much larger complexes and be able to look at them at much higher resolution,” Subramaniam says. “The atomic resolution structures we have been posting are still of fairly well-defined complexes. But when you go to more complex systems, we have only achieved lower resolutions.”
The key for the future will be to capture all of the movement and dynamics of protein complexes, without losing resolution. The group will use methods they pioneered over the last decade to image whole cells at high resolution to better understand how these complexes function in the context of the whole cell.
So far, the NIH team is doing what it can to stay at the forefront of a very crowded and growing area of research.
“It’s just taken off,” Subramaniam enthuses. “There’s exponential growth in the field. Until 2013…it was really a niche field.” And now? “There’s a real buzz across many disciplines that this could be a very powerful addition to the biologist’s toolkit.”