Visualizing the Conformational Dynamics of Proteins by Time-Resolved Electron Microscopy

Project Information

ProteinDynamics

Grant agreement ID: 759145

DOI

10.3030/759145

Project closed

EC signature date 30 August 2017

Start date 1 March 2018

End date 31 August 2023

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 2 000 000,00

EU contribution

€ 2 000 000,00

Coordinated by

ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE
Switzerland

Periodic Reporting for period 4 - ProteinDynamics (Visualizing the Conformational Dynamics of Proteins by Time-Resolved Electron Microscopy)

Reporting period: 2022-09-01 to 2023-08-31

Summary of the context and overall objectives of the project

Proteins make up the machinery of life and perform a multitude of tasks in living organisms. They are involved in harvesting energy from the environment, processing nutrients, sensing stimuli, or reproduction of the organism. Understanding the function of a protein may allow us to regulate it or combat dysfunction related to disease and is therefore crucial for biomedical applications. Much of our knowledge of proteins derives from atomic-resolution structures that are obtained with methods in structural biology, such as x-ray crystallography or cryo-electron microscopy. The structures such obtained are static, with available methods giving only limited information on the motions of proteins. However, by their very nature, proteins are dynamic systems, nanoscale machines that undergo a range of motions to perform their tasks. In the absence of direct observations of these dynamics, our understanding remains fundamentally incomplete. The goal of this project is to develop novel methods for imaging proteins while they perform their task, so as to obtain a movie of the processes involved. To give an example of a potential application – observing how the presence of a drug molecule alters the motions of a protein would significantly improve our understanding of the action of a drug and help researchers develop new types of medication.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The approach we are pursuing for watching the motions of proteins in real time involves high-speed observations with an electron microscope. While electron microscopes excel at recording atomic-resolution images of proteins, they are currently not fast enough to capture most protein motions, which typically occur on a timescale of microseconds to milliseconds. We have developed a new approach that overcomes these limitations by developing a microsecond time-resolved variant of cryo-electron microscopy. In cry-electron microscopy, the three-dimensional structure of proteins in determined by imaging them with an electron microscope, with the proteins embedded in so-called vitreous ice. This is a glassy state of ice that can be produced by rapidly cooling a solution of the proteins, so that the solution does not have time to crystallize. We have shown that we can use a laser beam to rapidly melt such a cryo sample. Once the sample is liquid, we can induce dynamics of the embedded proteins. As they unfold, we then switch the laser off that keeps the sample liquid, so that it cools rapidly and revitrifies, trapping the proteins in their transient states, in which we can subsequently image them. Importantly we can do so rapidly, with microsecond time resolution. Moreover, we have shown that we can obtain near-atomic resolution structures of the proteins with this approach.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Most recently, we have for the first time applied our approach to a real-life system, by studying the dynamics of the capsid of CCMV, a plant virus. Our work elucidates the intricate, microsecond motions of the capsid of the virus that are crucial for its viral life cycle and helps us understand the workings of this sophisticated nanoscale machine. Importantly, the methodology we have developed as part of the ERC grant is currently the only technique that allows one to observe these dynamics. Moreover, we believe that it will enable studies of the motions of a wide range of proteins. This promises to fundamentally advance our understanding of how proteins function.

Molecular models of the capsid of CCMV in its extended (left) and contracted state (right).