Summary:
UCLA researchers in the Department of Biological Chemistry and Physiology have developed a novel method to collect MicroED datasets using a direct electron detector.
Background:
Microcrystal electron diffraction (MicroED) is a method in electron cryo-microscopy (cryo-EM) that exploits the strong interaction of electrons with matter to determine high-resolution molecular structures from crystallized samples. This technique is particularly useful for samples that may be sensitive to the beam used in conventional X-ray crystallography, allowing damage-free structure elucidation to be completed. When this technique is used in conjunction with a transmission electron microscope (TEM), small molecule samples can have their structure determined with high accuracy directly from powders without crystallization: outperforming conventional X-ray crystallography. In MicroED, crystals are continuously rotated in the electron beam of a TEM, and a high-speed camera is used to record a shutterless movie of the resulting diffraction patterns. Direct electron detecting (DED) cameras are equipped on the majority of current TEMs in the cryo-EM field. However, it was previously thought that DED cameras were incompatible with this setup because of concerns over damage to the sensor by the intense incident beam as well as strong diffraction reflections. This has limited the practicality of MicroED as a technique because it required a dedicated camera that is not typically used in other cryo-EM modalities. Considering the multitude of benefits of MicroED, there exists a current need for a way to make the technique more approachable for widespread adoption.
Innovation:
UCLA researchers have used MicroED to collect essentially damage-free structure solution in a TEM while utilizing a DED camera. The researchers compared collected MicroED data from a series of microcrystals, using a DED and a conventional diffraction-optimized camera. Importantly, the DED was fitted with a thick scintillator to ensure the reliable capture of weak intensity Bragg spots. The researchers showed that a reliable structure solution was possible using the DED, and offered some unique advantages over the conventional diffraction-optimized camera setup. Notably, the DED setup allowed the collection of data at lower exposures in shorter time frames. This has immediate implications for efforts to automate MicroED data collection, where the efficient use of shared resources may be a significant concern, but also leads to structural models with reduced radiation damage. As a consequence, the researchers have provided a method to make MicroED more approachable to a wide range of researchers, potentially revolutionizing the field of structure determination.
Potential Applications:
• Higher resolution of MicroED structures
• Small molecules
• Natural products
• Drug discovery
Advantages:
• Increased practicality of MicroED technique due to it not needing a dedicated camera
• The ability for MicroED to be integrated as a shared resource with high-throughput capability
• Decreased amount of damage to the same due to low-radiation
• Femtogram amounts of material used for atomic resolution structures
Development to Date:
This method has been developed and optimized on proteinase K microcrystals.
Related Materials:
Hattne, J., Martynowycz, M., Gonen, T. MicroED with the Falcon III direct electron detector. IUCrJ. 6, 921-926 (2019)