Making time-resolved cryo-EM available for the community of structural biologists: validate, improve, derisk

Introduction

Knowledge of protein structure is fundamental for the understanding of biology, disease mechanisms, drug development, and biotechnology. Recent progress with electron cryogenic microscopy (cryo-EM) simplified and tremendously accelerated the determination of protein structures including multiple protein conformations.

Challenges in Protein Conformation Analysis

Many functionally important protein conformations and protein-protein complexes are transient and are not easily accessible by conventional cryo-EM. These transient states can, however, be freeze-trapped and their structures solved to high resolution using the time-resolved cryo-EM (trEM). Despite its long history and high interest from the cryo-EM community, technical difficulties prevented the spread of the methodology, and no commercial devices enabling trEM sample preparation exist.

Project Overview

During the ERC-CoG project, we developed a new approach to the preparation of trEM samples which has advantages over the other reported technologies. In this POC project, we propose to undertake steps that will explore the suitability of our technology for application to a wide variety of protein samples and to evaluate the commercial potential of the technology with the aim of bringing it to cryo-EM users rapidly.

Proposed Steps

To achieve this, we plan to:

1. Expand the number of protein samples to be tested on our instrument, thus enabling rapid user access and simultaneous validation of the methodology.
2. Perform additional research to further improve the method by addressing the remaining bottlenecks.
3. Make steps towards defining the best process and materials for industrial production of microfluidic chips.
4. Redesign the time-resolved plunger into a compact pre-industrial prototype.

Conclusion

These steps will broadly validate, improve, and derisk the technology to make it more attractive to cryo-EM instrumentation-making companies, facilitating the introduction of the technology to the cryo-EM community.