Periodic Reporting for period 1 - CoCoPro (high-Coherence, ultraCold electron diffraction for molecular movies of membrane Proteins)
Periodo di rendicontazione: 2022-09-15 al 2025-05-14
On a molecular level, all biological processes are carried out by proteins. Membrane proteins act as pumps and gates in the cell membrane, thus controlling all traffic that enters and leaves the cell. Because of this, they are important targets for medicine while their malfunctioning is at the heart of a plethora of diseases. But despite their enormous importance, membrane proteins are poorly understood as they are notoriously difficult to study using traditional methods based on X-rays. The more recently developed method of cryo-electron microscopy has revealed the structures of some of these proteins, but as it requires the sample to be frozen the obtained images are inherently static.
Now imagine that we could have an instrument that allows making movies of such proteins, so we can see exactly how they work and, also, why sometimes they do not work. Such a tool could revolutionize biochemistry, giving a unique insight in the structure-function relationship of nature’s molecular machinery, and helping scientists to understand membrane-protein-related diseases.
To make these molecular movies, I use a unique source that produces ultrashort flashes of electrons. Each of these flashes takes a snapshot of the protein, and together these snapshots form a molecular movie of the conformational change of the protein. To resolve the protein at high resolution while limiting radiation damage, the snapshots are taken in the form of diffraction patterns from 2D protein crystals. For this to work, the wave nature of the electrons needs to be very pronounced, i.e. the electron bunch needs a good coherence. To obtain such high-quality electron bunches, I use a unique approach: in a vacuum, Rubidium atoms are first laser-cooled to almost absolute zero temperature and then carefully ionized with a femtosecond laser pulse. The thus produced ultracold electron bunches are sufficiently coherent to create diffraction patterns from the protein crystal. In summary, by combining principles from fundamental physics with state-of-the-art technology and biochemistry, I aim to deliver a proof of concept that on the long term will advance medicine and other research fields through an improved understanding of membrane proteins.
Now imagine that we could have an instrument that allows making movies of such proteins, so we can see exactly how they work and, also, why sometimes they do not work. Such a tool could revolutionize biochemistry, giving a unique insight in the structure-function relationship of nature’s molecular machinery, and helping scientists to understand membrane-protein-related diseases.
To make these molecular movies, I use a unique source that produces ultrashort flashes of electrons. Each of these flashes takes a snapshot of the protein, and together these snapshots form a molecular movie of the conformational change of the protein. To resolve the protein at high resolution while limiting radiation damage, the snapshots are taken in the form of diffraction patterns from 2D protein crystals. For this to work, the wave nature of the electrons needs to be very pronounced, i.e. the electron bunch needs a good coherence. To obtain such high-quality electron bunches, I use a unique approach: in a vacuum, Rubidium atoms are first laser-cooled to almost absolute zero temperature and then carefully ionized with a femtosecond laser pulse. The thus produced ultracold electron bunches are sufficiently coherent to create diffraction patterns from the protein crystal. In summary, by combining principles from fundamental physics with state-of-the-art technology and biochemistry, I aim to deliver a proof of concept that on the long term will advance medicine and other research fields through an improved understanding of membrane proteins.
This project is based on instrumentation development. The project started with an electron source developed previously at TU Eindhoven.
At the start of the project several necessary upgrades the source were identified, all related to the transition of the setup from an experiment in itself towards a useful instrument.
A large part of the project was spent designing and implementing these upgrades, which included increasing the electron energy, improving the stability of the setup, and mitigation of the unwanted effects of magnetic fields on the electron beam.
Another main part of the project was spent on the preparation and characterization of protein samples, first on hydrophobin (HFBI) and then bacteriorhodopsin (aka purple membrane).
For the phasing of diffraction patterns of these 2D protein crystals, specific software is needed, of which a working version was obtained through collaboration.
Finally, an optical parametric amplifier (OPA) was built to generate synchronized laser pulses with a wavelength centered at the absorption peak of bacteriorhodopsin. This is a crucial step towards pump-probe experiments and the molecular movie.
Details can be found in the Technical Report (part B).
At the start of the project several necessary upgrades the source were identified, all related to the transition of the setup from an experiment in itself towards a useful instrument.
A large part of the project was spent designing and implementing these upgrades, which included increasing the electron energy, improving the stability of the setup, and mitigation of the unwanted effects of magnetic fields on the electron beam.
Another main part of the project was spent on the preparation and characterization of protein samples, first on hydrophobin (HFBI) and then bacteriorhodopsin (aka purple membrane).
For the phasing of diffraction patterns of these 2D protein crystals, specific software is needed, of which a working version was obtained through collaboration.
Finally, an optical parametric amplifier (OPA) was built to generate synchronized laser pulses with a wavelength centered at the absorption peak of bacteriorhodopsin. This is a crucial step towards pump-probe experiments and the molecular movie.
Details can be found in the Technical Report (part B).
The use of ultracold electrons is unique in the world, and its application to protein dynamics holds great promise. In this project important technological improvements have been made, two novel designs have been developed, two patent applications are ongoing, and a number of 'early adopters' have been identified. While some results of the project have been published in peer-review, other results require further research/development to determine the potential of the technique.
This research is currently continued at TU Eindhoven.
This research is currently continued at TU Eindhoven.