Cryo-EM breakthrough: New technique enables high-resolution imaging of small protein molecules

The 2017 Nobel Prize in Chemistry celebrated the groundbreaking cryo-electron microscopy (cryo-EM) technique, allowing atomic-level imaging of biological molecules. However, cryo-EM had limitations, mainly with smaller proteins.

UCLA biochemists, in collaboration with the pharmaceutical industry, devised a solution. They engineered a 20-nanometer cube-shaped protein scaffold with sturdy tripod-like protrusions to secure small proteins in place. During image processing, the scaffold could be digitally removed, leaving a precise 3D image of the small protein of interest.

This innovation is crucial for researching potential drugs targeting small to medium-sized proteins associated with challenging human diseases. It was successfully tested on a protein under investigation for potential cancer treatments. Scientists anticipate that this expansion of cryo-EM’s capabilities will enable them to pinpoint specific protein locations for therapeutic targeting.

This pioneering research has been published in the Proceedings of the National Academy of Sciences.

Cryo-EM functions by directing electrons through frozen material, capturing images of molecules, like proteins, as they exist in the sample. These images, taken from various angles, are processed by computers to create an accurate 3D representation, separating the background, grouping similar orientations, and producing high-resolution, 3D molecule images.

When dealing with the tiniest protein molecules, their size makes it challenging to determine their orientations in images, leading to lower resolution. Previous attempts involved attaching small molecules to larger scaffolds, but flexible attachments resulted in blurry images due to varying angles and orientations.

“The reason behind the blurry images,” explained Todd Yeates, UCLA’s distinguished professor emeritus of biochemistry and the paper’s corresponding author, “is the computer’s struggle to generate a clear composite image when it can’t accurately determine molecular orientations.”

An electron microscope image of scaffolds attached to the protein KRAS (background). The left circle highlights one imaging scaffold, the second displays the 3D structure of the imaging scaffold bound to KRAS, and the third shows a close-up of KRAS attached to the cancer drug AMG510. Credit: Proceedings of the National Academy of Sciences (2023). DOI: 10.1073/pnas.2305494120

In their recent study, the scientists ingeniously designed a scaffold with tripod-shaped protrusions, effectively immobilizing proteins and achieving the desired higher-resolution images.

Yeates elaborated on the concept: “By securely affixing the small molecules to larger scaffolds in a rigid manner, we created particles large enough for imaging, all sharing precisely the same 3D structure. From there, the standard process seamlessly constructed the high-resolution 3D image.”

Roger Castells-Graells, the lead author of the study and a UCLA postdoctoral researcher, revealed that they experimented with different scaffold shapes before settling on the version with tripod-shaped protrusions. He noted, “Initially, we tried a single outward ‘stick,’ which didn’t yield optimal results. The new scaffold, featuring triplets of protrusions that converge like tripods, firmly grips the protein.”

To validate their scaffold’s effectiveness, the researchers conducted experiments with a protein known as KRAS. KRAS plays a significant role in cell proliferation and is implicated in approximately 25% of human cancers. It holds immense interest for pharmaceutical researchers as pinpointing specific locations on the protein linked to its cancer-causing properties could pave the way for designing drugs that counteract those effects, potentially advancing cancer treatment.

Employing cryo-EM alongside their innovative scaffold, the UCLA-led team successfully visualized the atomic structure of KRAS bound to a drug molecule under investigation for potential lung cancer treatment. Their work demonstrated that this scaffolded cryo-EM approach can elucidate how drug molecules interact with and inhibit cellular proteins such as KRAS, offering valuable insights for developing more potent medications.

Castells-Graells emphasized that the applications of this groundbreaking technique extend beyond cancer drugs: “Our modular scaffold can be configured to capture and secure various small protein molecules, opening doors to a wide array of possibilities.”

Source: University of California, Los Angeles

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