Solving Protein Structures Made Simple

By Keith Gillogly

X-ray crystallography involves coaxing tiny proteins to form crystals large enough to be analyzed with X-rays — the delicate growing process can take months, even years. Alternatively, cryogenic electron microscopy works well for mapping large proteins but struggles with smaller ones, including most human proteins.

Now, a research team at the Jacobs School of Medicine and Biomedical Sciences has demonstrated how the technique microcrystal electron diffraction (MicroED) can quickly and efficiently create an extremely detailed 3D map of a protein using tiny crystals using widely available equipment. Creating precise protein maps is essential to developing new drugs and therapies. 

Led by Michael Martynowycz, PhD, assistant professor of structural biology at the Jacobs School, the team’s findings are described in a recent Nature Communications paper titled “Direct from the Seed: An Atomic Resolution Protein Structure by Ab Initio MicroED.”    

The group’s findings started with a combination of curiosity and happenstance. After struggling to find a supplier, the team obtained a sample of the protein crambin, whose structure has provided a benchmark for many studies.

The researchers then began breaking down the crambin seed into tiny proteins for further study. After suspending the sample in ethanol, Martynowycz figured they should check it for any precipitants. Using a light microscope, the team took a look and were surprised to find bunches of tiny nanocrystals sloughed off from the sample.

Instead of discarding these runoff nanocrystals, the researchers tried analyzing them with X-rays. But the X-rays destroyed the delicate nanocrystals. So, they thought they’d try using MicroED.

MicroED involves doing crystallography inside of an electron microscope. Electrons have mass and charge and interact strongly with matter, explains Martynowycz, who’s senior author on the paper. Figuring out their positioning can create incredibly precise molecular maps.

MicroED has existed for years, but the crystallography technique often requires using a similar protein as a reference or template. Here, the researchers achieved their results ab initio, meaning from scratch, and also didn’t have to painstakingly grow any crystals.

The nanocrystals diffracted to sub-angstrom resolution. “An angstrom is one-tenth of one nanometer; it’s smaller than the distance between two carbon atoms,” Martynowycz says.

At that level of detail, Martynowycz says, they could map every atom in the protein. 

Martynowycz hopes that other structural biologists and researchers will use this method for their own investigations. The code, methodology and steps behind it are all open access, he says.

“You can do this with any transmission electron microscope. You don’t need super high-end stuff,” Martynowycz says. The process can be completed in a day’s work, he adds.

Martynowycz credits his lab members with making these findings possible. He says that he’ll be relying on their multidisciplinary expertise for their next challenge: mapping protein charges using MicroED.

“Luckily the lab is full of different, spectacular people. We have computational people, chemists, physicists, biologists. And so, we have the broad expertise to try and figure this out,” he says.

Mapping out the positive and negative charges on a protein is critical to understanding how it will interact with other molecules and potential therapeutic agents.

“Charges basically dictate the whole game of bonding and interaction,” Martynowycz says. “So, how something bonds and how something binds and releases are entirely dictated by the charge landscape of the protein.”

For Martynowycz, resolving the molecular structure of crambin from scratch using MicroED is something of a full circle moment. Crambin was also the first protein resolved by the renowned mathematician and Nobel Prize laureate Dr. Herbert Hauptman using purely mathematical methods and no reference or template.

Hauptman’s name, of course, adorns the UB Hauptman-Woodward Research Institute, from which Martynowycz and his colleagues operate.   

Additional authors on the paper include first author Purna Chandra Rao Vasireddy, PhD; Devrim Acehan; Timothy Low-Beer; and Katherine A. Spoth, PhD, all of the Department of Structural Biology; and Matthew R. Crawley, PhD, of the Department of Chemistry at UB.