Periodic Reporting for period 2 - Ti-EM (Methodological developments for time-resolved single particle cryo-EM)
Reporting period: 2018-12-01 to 2020-05-31
The structures of the conformers involved in the functioning of molecular machines 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 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.
Gaining an understanding of the mechanisms of biological molecular machines is important because of their central role in human metabolism. Even small alterations in the efficiency of these macromolecules, due to, for instance, inherited or acquired mutations, or toxic substances, have a strong impact on the complete organism and cause diseases.
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. 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.
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 developed methods are validated on selected proteins including GroEL/ES complex and used to study mechanisms of the human ryanodine receptor, respiratory complex I and a ligand-gated ion channel.
Up to this time point, two setups to control flow and analyze processes taking place in microfluidics chips were built: one for time-resolved cryo-EM and other 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. Specialized software enabling user control over protein flow in the chip and cryo-EM sample preparation has been developed. We are working further to integrate all develop functionalities in one instrument and validate the potential of the instrument for miniaturized protein purification.
Significant progress has been achieved with the development of a microfluidic device for time-resolved cryo-EM. Microfluidic chips integrating miniaturized rapid mixer and droplet generator have been designed, manufactured and integrated with specially designed miniaturized cryo-plunger. The preparation of time-resolved cryo-EM samples is currently being validated with test samples. By the end of the project, we expect to develop the new methodology into a reliable technique enabling studies of a large range of temporary processes occurring with proteins in the cells and validate this approach on a set of selected proteins.
The project has also advanced with preparation and structural characterization of the proteins that will be used to assess the performance of the developed methodologies and apply the developed methods to understand the functional dynamics of the selected protein complexes. To this point procedures for protein production, purification and structural characterization using single-particle cryo-EM have been established and structures for most of the proteins were solved by cryo-EM in their resting states.
By the end of the project, we expect to establish procedures for time-resolved sample preparation for these proteins and resolve high-resolution conformations and kinetic of the transitions between the conformations using time-resolved cryo-EM.
The new approaches to time-resolved cryo-EM sample preparation significantly reduce sample consumption and will enable better reproducibility in the preparation of time-resolved cryo-EM grids by the end of the project. In the second half of the project, we expect to advance beyond the state of the art in time-resolution and in resolving higher-resolution conformations of the reaction intermediates in the selected molecular machines and obtain new insights into their mechanisms.