Vitrifying Biological Samples for Cryogenic Electron Microscopy

Within less than one decade, cryogenic electron microscopy (cryo-EM) has become an indispensable tool for structural biologists to study the structure and function of important biomolecules.

Individual macromolecular assemblies are imaged under cryogenic conditions within highly automated electron microscopes after which these images can be computationally combined to form a high-resolution view of key proteins and complexes.

Understanding the 3D structure of macromolecular complexes is important for the design of new drugs and vaccines: e.g., structural knowledge of the SARS-CoV-2 protein greatly aided the rapid development of COVID-19 vaccines.

In addition, structural knowledge of the two SARS-CoV-2 proteases as well as of virus replication complexes aided in the development of COVID-19 drugs.

With the support of NWO, we produced a fascinating animation showing all molecular players that play a role in the lifecycle of SARS-CoV-2: https://youtu.be/XlOi2hVGtg0.

The animation incorporates numerous experimental 3D structures of macromolecular complexes in isolation as well in cellular context. Being able to obtain these structures requires optimal sample preparation procedures. Sample preparation is an indispensable step for cryo-EM.

Previously, we developed and patented the technology to prepare purified protein samples for cryo-EM by using jets of cryogenic fluid directed onto the sample.

We implemented this technology into a device, the VitroJet, which has been developed into a commercial product by our startup CryoSol-World B.V., and has been sold worldwide. More recently, we filed an extra patent, protecting a technique to enable us to vitrify thicker samples.

We expect in situ structural biology, where macromolecular assemblies are studied within the interior of a focused-ion-beam milled frozen cell, to become the next revolution in structural biology. Vitrifying cells, rather than thin layers of isolated proteins in solution, comes with specific challenges.

Cell vitrification requires higher cooling rates, a different temperature control, and alternative sample carriers to ensure a smooth connection to the subsequent milling and imaging steps.

Over the last four years, we went through the discovery phase (TRL 1, 2 and 3) and touched upon many aspects of the development phase.

We now wish to develop, validate and demonstrate a new prototype, fully dedicated to cell vitrification: the VitroJet4Cells.

While CryoSol-World focuses on a high-end machine to prepare samples of purified proteins, we seek out to launch a new start-up to bring the VitroJet4Cells through the next TLR-phases such that this new device could make full impact on the community as well.