Smart EPU Software collection methods for challenging cryo-EM samples

Challenging cryo-EM sample collection

Screening challenging cryo-EM samples such as the COVID-19 spike protein can present significant difficulties for researchers. A study published in Nature Communications highlights the arduous task of investigating the SARS-CoV-2 spike protein, which is further complicated by issues like preferred orientation and the absence of detergents.

As new variants of challenging sample types continue to emerge, each bearing complex combinations of non-synonymous mutations that contribute to increased transmissibility, understanding how these mutations impact the conformational state of key proteins becomes crucial. Accurate determination of these impacts necessitates a precise understanding of the biophysical tools and methods commonly employed to investigate such proteins.

The last thing researchers need is wasted time, effort, and the potential loss of valuable samples due to inadequate screening and collection tools. The stakes are high, and the demand for reliable and efficient methods is paramount.

Grid screening for single particle analysis

During the screening of challenging samples, optimal grids and sample conditions are critical for subsequent high-resolution data collection. To address these challenges, the combination of the Thermo Scientific™ VitroEase™ Buffer Screening Kit and Thermo Scientific Smart EPU Software with Multigrid offers a robust solution. Additionally, Smart EPU Software’s tilted data collection using aberration-free image shift significantly increases image acquisition throughput.

To overcome the issues associated with the SARS-CoV-2 spike protein, researchers added a detergent to their sample preparation. The VitroEase Buffer Screening Kit, which includes various types of detergents, was employed. Researchers prepared 12 grids in total using six different protein-detergent mixtures at two different concentrations. Additionally, the Multigrid was also used for high-resolution acquisition, allowing researchers to acquire two full datasets using two different concentrations of the detergent.

Using the EPU Multigrid Software, researchers were able to conduct multiple data acquisition experiments within a single day. The data from these experiments were analyzed using CryoSPARC Live™ Software, which enabled the identification of the most effective detergent.

Structural determination of the SARS-C cryo EM sample

Structural determination of SARS-C. Image adapted from Egri, SB, et al. under CC BY 4.0.

Currently, Smart EPU Software with Embedded CryoSPARC Live offers several key benefits, including the ability to run multiple data acquisition experiments fully unattended, i.e., overnight or during weekends, thereby maximizing efficiency. It significantly reduces microscope idle time and eliminates the need for manual grid switching during off-hours, favoring better utilization of human resources. Additionally, the software accelerates the setup and scheduling of single-particle analysis screening sessions with minimal manual interventions and a low degree of user expertise. The screening session execution can also be conducted unattended, leading to focus data acquisition in the best grid, best grid areas, and optimal imaging conditions for the sample under scrutiny. Overall, Smart EPU Software facilitates the creation of new and more efficient grid screening workflows, enhancing the overall screening and high-resolution acquisition process.

Results of cryo-EM sample screening with EPU Multigrid Software

The implementation of EPU Multigrid Software in combination with VitroEase buffers resulted in:

• The screening of six protein-detergent mixtures in two concentrations (12 grids in total) for cryo-EM structure determination completed in one day.
• Two high-resolution data collections scheduled for two concentrations of the selected detergent.

Cryo EM Discovery Viewer helps to precisely determine where specific images were captured on a cryo EM sample during the single particle analysis

Discovery Viewer, a navigation tool specific for cryo-EM, is an add-on that helps to precisely determine where specific images were captured on a sample during the single particle analysis workflow.

Conclusions

For the data collection and screening of the most challenging samples, the combination of Smart EPU Software and the VitroEase Buffer Screening Kit can significantly simplify and expedite screening and data acquisition sessions. This method provides researchers with the confidence that they are obtaining accurate and reproducible results.

The use of these advanced and innovative tools aims to increase efficiency, productivity, and ease of use for the cryo-EM workflow, ensuring that researchers can focus on their scientific inquiries without the hindrance of technical obstacles.

Resources

VitroEase Buffer Screening Kit application note >>

Smart EPU Software datasheet >>

Learn more at thermofisher.com/smart-epu >>

CryoSPARC Live is a trademark of Structura Biotechnology Inc. and is used under license.