Obtaining a tilt series in a TEM currently takes 20-60 minutes which slows down the cryo-ET workflow considerably. Fortunately, next-generation direct detectors, hardware for faster data transfer as well as methods for rapid tilt series are currently being developed . Combined, a tilt series could be obtained in only a fraction of the time it currently takes, speeding up the workflow considerably. This development also increases the need for easy and automatic sample preparation methods to optimally make use of the cryo-TEM. Automatic sample preparation combined with high-speed TEM acquisition will result in the possibility to resolve large amounts of biological structures.
As more and more structures become available data processing could be optimized by automatic mapping. A tomogram that contains several biological structures could be automatically screened against a database. Known structures would be fitted to the structures visible in the tomogram, showing which known structures are present. Routinely obtaining biological structures could also result in the rise of quantitative structural biology: doing statistical analysis to gain a deeper understanding of structure-to-structure variations.
Cryo-ET will reveal structural information of many cellular processes and pathways
A major step forward has been made possible by cryo-ET by moving from obtaining structural data in purified samples to getting the same high resolution data in their biological context, or in situ. At the moment, many studies are focusing on large protein complexes that “stand out of the crowd” due to their known size or location, making them relatively easier to identify and image. As the cryo-ET technology develops further, it will become easier to identify increasingly rare cellular structures and events. Obtaining high resolution structural data of rare cellular events will have a great impact on fields such as drug development and cellular immunology, but will also likely reshape our knowledge on many of the textbook signalling pathways and trafficking routes of the cell.
Even though proteins often assemble in stable multiprotein complexes, most protein-protein interactions are transient in nature . These interactions, often accompanied by changes in protein fold or shape, hold crucial information on how and where important processes take place. Capturing the structural detail of such transient interactions in their cellular context seems like an unobtainable feat at the moment, but with the current advancements in cryo-ET technology, it might not be that far away.
Likewise in the field of nucleic acid research, cryo-ET can play a fundamental role unravelling processes such as the post-transcriptional regulation by (non-coding) RNA. The change in secondary structure of RNA can directly translate into a specific function, such as regulation of gene expression, protein interaction or RNA-RNA interaction . Since the first discoveries of non-coding RNA molecules, it has become apparent that RNA molecules regulate a host of complex cellular processes that are tied strongly to their confirmation . Here, cryo-ET could shed further light on the structure-function relation of RNA in its cellular context. Here the challenge lies in the vast amount of content in the cytoplasm and the relatively small size of signalling RNA molecules. For this, increased precision in finding molecules of interest with cryo-FLM and increased signal-to-noise ratio to resolve structures in the crowded cytoplasm are needed.
At DELMIC we strongly believe that cryo-ET will revolutionize the field of structural biology and become the method of choice to obtain the 3D structure of a biomolecule. We aim to make cryo-ET more accessible to researchers by providing solutions that simplify the workflow. We do so by consolidating the sample preparation workflow with an automated approach.
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