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Methodological developments for time-resolved single particle cryo-EM

Periodic Reporting for period 4 - Ti-EM (Methodological developments for time-resolved single particle cryo-EM)

Reporting period: 2021-12-01 to 2022-11-30

Major vital processes in living organisms are fulfilled by large protein or protein/nucleic acid complexes with a molecular weight of hundreds of kilodaltons to megadaltons. These include the synthesis and processing of polymers: proteins, DNA, and RNA; cellular transport, muscular contraction, cell division, and energy metabolism. Many of these complexes function as molecular machines converting the energy of a fuel, usually ATP, into mechanical movements and useful work. Walking of kinesin or dynein on microtubules, contraction of actin-myosin fibers, ratcheting of the ribosome, or rotary mechanism of ATP-synthase are spectacular examples of the conformational changes reminiscent of human-made machines and mechanisms. The apparent analogy between nano-machines and human-made machines is misleading, however. Nano-machines are essentially Brownian machines. In contrast to human-engineered machines molecular machines do not rely on gravity and inertia or materials as rigid as steel, they operate in a world with high viscosity and are found in constant thermal motion. When considered at the atomic level, molecular machines do not have an apparent directionality, yet they are fast, reliable, and often operate with nearly 100% efficiency. The conformational changes in the proteins perform a stochastic walk on a potential energy surface, where local minima define conformational states and transitions between the states are governed by thermal fluctuations. Importantly, the energy input in molecular machines is used to modify the potential energy landscape and provide specific directions for molecular motions.

On the other hand, the development of synthetic molecular machines is an important research field in chemistry and material engineering. The molecular-scale switches, motors, and robots promise to enable controlling phenomena occurring on the nanoscale with high accuracy, which is still very difficult today. Such molecular devices have the potential for many applications ranging from smart materials to accurate control of chemical reactions to applications in medicine where nano-robots targeting specific cell types and executing programmed operations could treat diseases. Human-engineered molecular machines, however, are still very simple in comparison to biological molecular machines. The widespread use of molecular machines in biology has long suggested that great rewards could come from bridging the gap between synthetic molecular systems and the machines of the macroscopic world. A detailed understanding of the mechanisms of biological molecular machines will provide examples of engineering principles used by nature, which can be translated into human-made molecular machines.
The structures of the conformers involved in the functioning of molecular machines need to be known to understand their mechanisms, and they can be determined using time-resolved cryo-EM. With this technique, the biological reaction of synchronized proteins is arrested by freeze-trapping at specified delays after reaction initiation. Next, the single-particle cryo-EM method is used to visualize individual protein complexes in vitreous ice, and to calculate the 3D reconstruction of the protein structure by applying averaging and 3D reconstruction techniques. The bottleneck of this approach is associated with sample preparation methods that are not mature enough to be applied to most of the protein sampled due to large sample consumption and poor reproducibility.

This project addresses the problem of sample preparation for time-resolved cryo-EM.
The aim of this project is to develop novel approaches to SP cryo-EM sample preparation based on microfluidics that 1) allow for solving high-resolution structures of proteins from thousands to million times less sample than used today and 2) allows freeze-trapping transient intermediates of the protein conformations with millisecond time resolution using small volumes of protein solution.
The project has two pillars: the development of new microfluidics-based tools for cryo-EM and the application of these tools to answer questions in structural biology.

We performed research on developing microfluidics-based methods for time-resolved cryo-EM and miniaturized protein purification and preparation of cryo-EM samples. This involved experimenting with various approaches of liquid mixing on microscopic scales, generation of stable and reproducible microjets, and constructing microliter and sub-microliter volume chromatographic columns.
Two microfluidics-based instruments were built as a result of the project. The first one enables the preparation of time-resolved cryo-EM grids reproducibly with 5 milliseconds time resolution using small amount of protein solution. It is based on a combination of droplet-based microfluidics mixer and laser-induced on-demand generation of jets of microdroplets. The second device was developed for protein micro-purification. Microfluidic chips have been developed for two-step protein purification and preparation of cryo-EM samples by blotless method consuming only a few nanoliters of sample per grid. It allows to use of only a few microliters of starting material, or even a single Escherichia coli colony to prepare high-quality cryo-EM grids from protein purified in this setup. Specialized software enabling user control over protein flow in the chip and cryo-EM sample preparation has been developed.

These methods have been validated using test proteins including apoferritin, beta-galactosidase, and respiratory complex I and GroEL:GroES chaperonin system. We showed that time-resolved experiments on GroEL:GroES systems unravel the kinetics of the complex formation, which has not been previously observed using a structural approach. Using a micro purification instrument, we obtained a 2.5 Å 3D reconstruction of beta-galactosidase expressed in E. coli cells and purified from only a few microliters of cell extract.
Both technological developments in micro-purification for cryo-EM and time-resolved cryo-EM grid preparation achieve results going beyond the state of the art. They will allow the preparation of cryo-EM grids starting with significantly smaller volumes of cell extracts than the state-of-the-art. This methodology accelerates the purification process and reduces the losses of the protein.
The new approaches to time-resolved cryo-EM sample preparation significantly reduce sample consumption and enable better reproducibility in the preparation of time-resolved cryo-EM grids and achieve higher time resolution. Which also goes beyond the state of the art.
The images summaries the device for time-resolved cryo-EM developed as the main objective o the ERC