After decades of frustration, scientists have successfully refined cryogenic electron microscopy (cryo-EM) by utilizing ultracold liquid helium to achieve remarkably high-resolution images of protein structures. This breakthrough overcomes a long-standing issue that paradoxically resulted in fuzzier images when using liquid helium compared to the warmer liquid nitrogen temperatures. The findings pave the way for electron microscopes with significantly enhanced clarity, promising new insights into a wide array of proteins, including crucial cell surface proteins that are key targets for drug development.
The fundamental principle of cryo-EM involves bombarding frozen protein samples with a powerful electron beam and analyzing how the electrons scatter off the atoms to reconstruct a 3D map of the protein’s atomic arrangement. While the electron beam is essential for imaging, it also causes radiation damage that can obliterate the protein within seconds. Freezing samples in liquid nitrogen (at 77 Kelvin) has been a crucial step in mitigating this damage, reducing it fivefold compared to room temperature and significantly improving image resolution. The theoretical expectation was that cooling further to liquid helium temperatures (around 4 Kelvin) would reduce radiation damage by another twofold, leading to even sharper images. However, this was not the case, with helium-cooled microscopes consistently producing inferior, fuzzier results, leading to the near abandonment of the technique.
Researchers have now identified the root cause of this perplexing problem: the ultracold temperatures cause the ice surrounding the embedded protein molecules to expand and buckle. This subtle distortion of the ice matrix pushes the protein molecules slightly apart, effectively blurring the resulting electron microscopy images. The long-held assumption that colder temperatures would simply shrink the ice and compress the protein molecules was proven incorrect.
To pinpoint the cause of the image degradation, the researchers resurrected a 20-year-old liquid helium-cooled electron microscope in their laboratory. They replaced protein samples with gold nanoparticles, which are strong electron scatterers, allowing for precise tracking of their positions down to the angstrom level (one-tenth of a nanometer). Imaging these nanoparticles at both 77 K and 13 K revealed that at the colder temperature, the ice expanded, causing the nanoparticles to move slightly further apart, confirming the buckling phenomenon.
The team engineered a new sample stage for the microscope to counteract this ice buckling due to liquid helium. They replaced the copper in the original stage with gold, which effectively conducts away stray electrons that can contribute to repulsive forces at the edges of the sample wells. They also significantly reduced the size of the holes in the stage, from 400 nanometers to just 100 nanometers. This miniaturization drastically reduced the volume of ice in each well, further restricting unwanted movements and stabilizing the sample.
The researchers then tested their modified stage with proteins of known structures. Images taken at both 77 K and 13 K showed a significant 1.5-fold improvement in resolution with the ultracold liquid helium cooling. This substantial enhancement promises to unlock finer details in protein structures and potentially enable the imaging of proteins roughly half the size of what is currently possible with cryo-EM. This includes challenging targets like cell membrane proteins, which are critical for drug development but difficult to study with traditional X-ray crystallography. The ability to visualize these “gatekeeper” proteins with greater clarity could significantly accelerate the discovery of novel medicines and enhance the training data for AI-driven protein structure prediction.
The cryo-EM community is already responding to this breakthrough, with microscope manufacturers actively working to incorporate helium cooling capabilities into their instruments, ushering in a new era of high-resolution structural biology.
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