Blobfish Breakthrough: New CryoPRISM Technique Reveals Cellular Structures in Detail

A new technique called cryoPRISM is offering biologists a clearer view of molecular structures within cells, potentially resolving a long-standing challenge in structural biology. Like realizing the blobfish appears “blobby” only when removed from the immense pressure of its deep-sea habitat, scientists have found that extracting biomolecules from cells to study them can alter their natural state. CryoPRISM aims to bridge this gap, allowing for visualization of these complexes with minimal disruption to their cellular environment.

Preserving the Cellular Context: A Delicate Balance

Structural biologists strive to understand how molecules function within cells, a process that hinges on determining their precise structure. Traditionally, this involved isolating biomolecular complexes for imaging. While this approach yields high-quality images, it risks distorting the molecules’ natural arrangement. Conversely, studying molecules in situ – directly within the cell – is technically demanding. CryoPRISM, developed by researchers at MIT, offers a compromise, capturing molecular structures in cells immediately after they’ve been gently broken open. This method, recently published in PNAS, preserves more of the natural interactions between molecules than traditional purification methods, while still providing sufficient resolution for detailed analysis.

Joey Davis, associate professor of biology at MIT and the faculty lead of the study, explains that cryoPRISM represents a “sweet spot,” preserving crucial cellular contacts while maintaining the resolution needed to observe molecular details. Even in well-studied systems like bacterial translation, the technique is revealing previously unnoticed molecular states.

An Unexpected Discovery: From Control to Breakthrough

The development of cryoPRISM stemmed from an unexpected observation during a separate research project. Mira May, a graduate student and co-first author of the study, was investigating ribosomal regulation in bacteria. Ribosomes are essential cellular machines responsible for protein synthesis, guided by instructions from mRNA. Cells regulate ribosome activity using additional proteins that alter their shape and function. May initially attempted to isolate ribosomes with their regulators using cryo-electron microscopy (cryoEM), a technique involving rapid freezing and imaging of molecules.

As a control, May prepared a sample containing unpurified bacterial lysate – essentially, everything released from burst cells. Expecting poor-quality images, she was surprised to find intact ribosomes interacting with their natural partners. This unexpected result quickly shifted the focus of the project from identifying regulators to developing cryoPRISM as a new method for visualizing cellular structures.

Uncovering Novel Ribosomal Biology

Once validated, cryoPRISM allowed researchers to identify ribosomal states previously undetected. One particularly intriguing finding relates to the behavior of ribosomes under stress. When bacterial cells encounter unfavorable conditions, like cold temperatures, they reduce protein synthesis. Ribosomes enter a dormant state, blocked by a hibernation factor called RaiA, preventing premature reactivation.

May’s team observed that some inactive ribosomes weren’t just interacting with RaiA, but also with an elongation factor called EF-G – a protein previously thought to only interact with active ribosomes. This observation mirrors findings in more complex organisms and suggests that the evolutionary origins of this interaction may be older than previously believed. The researchers propose that elongation factors may bind to hibernating ribosomes to protect both themselves and the ribosomes from degradation during stressful periods, essentially acting as a form of “short-term storage.” An unstressed cell might eliminate inactive ribosomes quickly, but a cell facing temporary stress may prefer to preserve them for rapid reactivation when conditions improve.

Implications for Understanding Evolution and Cellular Stress

This discovery has implications for our understanding of how cells adapt to environmental changes and how translation regulation evolved. The finding that bacterial ribosomes exhibit a similar protective mechanism to those in more complex organisms suggests a conserved evolutionary strategy for managing cellular resources under stress. The blobfish example illustrates how context is crucial; similarly, cryoPRISM highlights the importance of preserving the cellular environment when studying molecular interactions.

Expanding the Applications of CryoPRISM

May has already begun collaborating with other MIT researchers to apply cryoPRISM to challenging biological systems. This includes studying pathogenic organisms, which can be difficult to culture in large quantities for traditional purification methods, and analyzing red blood cells isolated from patients, which cannot be cultured at all. Beyond translation research, cryoPRISM represents a step towards the broader goal of structural biology: studying biomolecules in their natural cellular context. As Davis notes, the technique aligns with the growing trend in structural biology of moving “closer and closer to cellular context.”

Future Directions and Ongoing Research

The development of cryoPRISM is an ongoing process. Researchers are continually refining the technique to improve resolution and expand its applicability to a wider range of biological systems. Further studies are needed to fully elucidate the mechanisms underlying the interactions between elongation factors and hibernating ribosomes, and to explore the broader implications of this finding for cellular stress response and evolution. The team is also working to automate aspects of the cryoPRISM workflow to increase throughput and make the technique more accessible to researchers worldwide. This includes exploring new data analysis pipelines to efficiently process the large datasets generated by cryoEM imaging.

cryoPRISM promises to provide a more accurate and comprehensive understanding of cellular processes, paving the way for new discoveries in biology and medicine. For more information on cryo-electron microscopy and structural biology, resources are available from the National Center for Microscopy and Imaging Research.