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

Periodic Reporting for period 2 - ProteinDynamics (Visualizing the Conformational Dynamics of Proteins by Time-Resolved Electron Microscopy)

Reporting period: 2019-09-01 to 2021-02-28

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.
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 protein motions, which typically occur on a timescale of microseconds to milliseconds. Our approach to making this possible is to generate short and bright flashes of electrons, short enough to capture a crisp image of a protein during its fast movement and bright enough to obtain an image with sufficient contrast. In the last year, we have established a method for generating such electron pulses. It involves briefly heating a so-called Schottky emitter, a type high-brightness electron source, to high temperatures by illuminating it with a laser pulse. This heats the tip, from which electron emission occurs, to high temperatures, significantly higher than the emitter would ordinarily be able to withstand, and thus boosts electron emission for a duration of microseconds to milliseconds. We have shown that the electron pulses we thus obtain are able to capture atomic-resolution snapshots of the dynamics of nanoscale objects as they undergo rapid transformations and that these snapshots provide sufficient contrast to image proteins. We have thus established a key ingredient for our endeavor. Moreover, the technology we have developed opens up a range of new possibilities for high-speed observations of atomic-scale transformations.
With the necessary technological progress achieved, we have now begun studies of the dynamics of proteins. To this end we enclose a thin layer of water containing the proteins within a microchip with thin viewing windows and place the chip inside the electron microscope. We then use a short laser pulse to initiate the dynamics of the proteins and at a given point in time, take a snapshot with an intense, microsecond electron pulse. We then repeat the process to take images at different times, so as to stitch together the frames of a movie. By the end of the project, we hope to establish that acquiring such movies is possible for a range of different protein motions. Moreover, we want to apply existing computational tools to obtain the three-dimensional structure of the proteins as a function of time.