Once a high-quality protein sample has been obtained, there are still a few obstacles to overcome. One of the key challenges is preparing an optimal cryo-EM grid, where the protein sample is evenly distributed across the grid holes with maximal angular diversity. To streamline this optimization process, we can leverage the PUXANO cryo-EM grid portfolio—a collection of proprietary cryo-EM grids developed by our nanoengineers to address specific issues in cryo-sample preparation.

Some of the challenges we can resolve include preferred particle orientation, handling protein concentrations below 0.2 mg/ml, and preventing protein complex disassembly. Because of this, our technology enables access to protein samples that are typically challenging with standard grid methods.

During the screening phase, we will apply your sample to various grid types and use a medium-end microscope to evaluate which conditions and grid types yield the best results. Thanks to our advanced cryo-EM grid hardware, we have precise control over factors that influence particle behavior, allowing us to limit the screening process to just one iteration.

The screening process includes several grid variations, and for the best-performing grid, we will collect data overnight. This allows us to process the data early, yielding 2D class averages or a preliminary medium-resolution 3D density map.

The optimal sample conditions and grid type identified during grid screening are then used for high-end data collection. Typically, we allocate 1-2 days to obtain a high-resolution protein structure. The data is processed using a combination of open-source and proprietary single-particle image processing protocols. The resulting density map is used to build a protein model, allowing us to accurately position the atoms of the protein sequence and identify interaction interfaces. Specific applications include generating protein or protein complex models, epitope mapping, and identifying small-molecule interaction sites.

Several types of nanoparticles can be analyzed using cryoEM, including lipid nanoparticles (LNPs), virus-like particles (VLPs), vesicles, and exosomes. Within PUXANO, we have extensive experience analyzing these nanoparticle types using cryoEM and complementary orthogonal techniques. Specific qualitative information we can obtain includes particle morphology, DNA/mRNA load, size, and more. In addition, by analyzing large datasets, we can perform quantitative analyses to estimate particle size and class distribution. To support this, we have developed proprietary software that automates the analysis, providing robust statistical insights. For a high volume of samples, we can also employ other orthogonal techniques to increase throughput and validate scientific findings. Furthermore, thanks to our proprietary Nanoparticle Grid, we can overcome the biases often introduced during cryoEM sample preparation. Unlike conventional grids, where particles are pushed into or excluded from thicker ice regions, the PUXANO Nanoparticle Grid ensures even nanoparticle distribution, minimizing sample bias.