The best of both worlds
Imaging of radiosensitive materials with serial electron diffraction
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As proteins are the building blocks of life, knowledge of their inner structure is highly relevant for biological research. However, this task is complicated by the delicate nature of these materials. When exposed to X-rays – which are usually used to determine tiny structures – they suffer irreversible damage from the high-energy radiation. One possible solution is offered by facilities such as the European XFEL: Short and intense X-ray laser pulses allow femtosecond diffraction snapshots of nanocrystals grown from biological molecules, thereby minimizing the exposure time. By recording snapshots from thousands of nanocrystals (serial diffraction), a full image of the molecular structure can be reconstructed. However, time slots at these facilities are costly and scarce, which motivates the search for cheaper and more easily accessible techniques.
As an alternative to X-rays, electrons can be used for diffraction images. Usually, nanocrystals are rotated under the beam in an electron microscope to obtain the three-dimensional structure by “looking from all directions” – for proteins, however, that would mean too high of a radiation dose once again. In an attempt to solve the radiation damage problem, a team of researchers from Germany and Canada has now joined the serial diffraction approach from XFEL and the gentle radiation inside an electron microscope into a technique called serial electron diffraction.
Experimental setup
The researchers tested their technique on granulovirus particles, which had previously been studied at the European XFEL, and hen egg-white lysozyme crystals, whose structure is known from X-ray and rotation electron diffraction experiments. The nanocrystals were dispersed onto a thin carbon film and placed in a transmission electron microscope equipped with a LAMBDA-EM 750k camera, the prototype of our AMBER 750k camera. In a first step, the nanocrystals were mapped by scanning the grid with a low dose. In a second step, an electron beam of 100 nm diameter was automatically steered to the respective positions and a series of diffraction images of the individual nanocrystals were taken. “Besides the impressive speed and dynamic range of the LAMBDA detector, its large array size proved to be critical for achieving high-resolution structures of large objects”, explained Robert Bücker, lead author of the study.
The idea behind serial electron diffraction.
© Robert Bücker, with kind approval
| Setup | Philips Tecnai F20 TWIN S/TEM equipped with an AMBER 750k prototype |
|---|---|
| Camera | AMBER 750k Si prototype |
| Resolution | 786,432 pixels with 55 μm |
| Aquisition frequency | 500 Hz |
| Electron energy | 200 kV |
Results
The detector was acquiring 500 images per second, whereby one nanocrystal was exposed for 10 frames. The scientists thus obtained a short film for each crystal, enabling them to witness the melting away of the proteins under the increasing radiation dose. Nevertheless, the researchers could retrieve the protein structure from their large dataset using serial reconstruction methods that have been developed for use with XFEL data collection protocols, now serving dual purpose for use of electron sources. Their results compare well to and in parts improve on those obtained with different techniques, which suggests that serial electron microscopy is a viable alternative to current protein crystallography techniques.
Diffraction pattern (left) from one out of thousands of nanocrystals (top right). The bottom right panel shows the nanocrystal under investigation, whereas the red circle corresponds to the diffraction nanobeam diameter and the colored lines indicate the lattice vectors.
© Robert Bücker, with kind approval (truncated)
References
Bücker, R., Hogan-Lamarre, P., Mehrabi, P. et al. Serial protein crystallography in an electron microscope. Nat Commun 11, 996 (2020). https://doi.org/10.1038/s41467-020-14793-0
R. Bücker, P. Hogan-Lamarre, and R. J. D. Miller, “Serial Electron Diffraction Data Processing With diffractem and CrystFEL,” Front. Mol. Biosci., vol. 8, 2021, doi: 10.3389/fmolb.2021.624264
This information illustrates a real-world application of an AMBER 750k prototype, developed and manufactured by X-Spectrum. We gratefully acknowledge the voluntary support by the mentioned scientist.