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Developed in late 2013, Microcrystal Electron Diffraction (microED) is a promising technique for Structural Biology and structure-based drug design. Using a standard, 200-300 keV Transmission Electron Microscope (TEM) in combination with a hybrid-pixel direct detector, microED can deliver high-quality crystallographic data for samples that don’t form large and stable crystals. What are the differences between microED and its older brother, X-ray Diffraction (XRD), and why is it crucial to develop dedicated instruments for microED? In this blog, we’ve summarized all you need to know.
Diffraction pattern collected with DECTRIS QUADRO detector using microED technique.
In simple terms, “diffraction” refers to various phenomena that occur when a wave interacts with an object, giving rise to interference patterns. If the subject of the analysis is a crystal consisting of atoms or molecules that are organized in a periodic fashion, the resulting diffraction pattern can be used to reconstruct the crystal’s microscopic structure. Today, there are a lot of techniques based on this phenomenon, and they are used to study a variety of systems: from solid-state materials such as metals and semiconductors to biological systems such as proteins, viruses, and pharmaceutical compounds. The most widespread techniques are single-crystal X-ray Diffraction (XRD) and Powder X-ray Diffraction (PXRD).
Single-crystal XRD is a mature technique that provides the most detailed structures, but it requires macroscopic, high-quality crystals in order to deliver usable data. Since growing such crystals is often a time-consuming and challenging process, sample preparation is the technique’s most significant bottleneck—not to mention that some molecules do not always form large crystals and therefore cannot be investigated using this method.
In that case, PXRD provides an alternative route for structure determination, since it relaxes the requirement for large crystals. It uses powder (a mix of microscopic crystals), but it is much less efficient than XRD.
This is the point where Microcrystal Electron Diffraction (microED) comes into play.
Up until 2013, when microED was introduced by a group led by T. Gonen, many microcrystalline materials were dismissively qualified as powders. However, given crystals’ dimensions that range from a few tens to a few hundred nanometers, these are the perfect target for microED.
Since the interaction of electrons with materials is much stronger, one needs much less diffracting material in order to obtain good reflections. Thus, microED is a complementary technique that has the potential to fill the gap between single-crystal XRD and PXRD. It brings a “new era for biological structure determination” 2, owing to the greater diversity of samples that can be measured by using fewer and smaller crystals.1, 3
MicroED can generally be done using a typical, 200-300 keV Transmission Electron Microscope (TEM). However, a few microED studies have also been published by several groups who used lower energy ranges (e.g. the Gemmi group in Pisa).
In these instruments, hundreds of crystals can be deposited easily onto a standard, electron-transparent TEM grid, and the screening process can be automated to some extent. Once a good microcrystal is found, the actual data collection may take only a few seconds when using hybrid-pixel detectors—thanks to their high frame rate and high dynamic range. This combination of a high-energy electron beam and a fast-counting detector offers excellent spatial resolution and a high throughput.
At this point in time, many protein structures have been solved successfully using microED, and improvements in the methods of analysis and the refinement algorithms have enhanced the resolution and accuracy of the technique. Also, the development of dedicated instruments for microED promises a significant step forward in this field.
To improve the quality of data that are obtained using microED and fully enable its application potential in pharmaceutical and chemical laboratories, a specially designed tool with a high degree of automation and software integration is required. Of course, many of the necessary functionalities are already available on a traditional TEM; however, to assure ease of use and a high throughput, several companies are investing resources to develop dedicated electron diffractometers.
Eldico Scientific and Rigaku are leading the way in this effort, but the academic community is also highly involved in the process: Grüne and collaborators, for example, contributed defining design guidelines for such instruments. These include, but are not limited to: improvements in sample illumination; high-precision goniometers; integrated software; and noise-free, fast detectors.
Hybrid-pixel electron detectors are capable of single-electron counting. Therefore, even for delicate samples that require the incident beam’s intensity to be particularly low, you can be sure to capture every diffraction event. These detectors also feature an extremely fast frame rate (up to 2,000 frames per second), shutterless operation, a high dynamic range, and radiation hardness.
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