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What is microcrystal electron diffraction?
MicroED is an exciting new technique that allows fast, high-resolution structural determination of small molecules and proteins. Atomic details can be extracted from individual nanocrystals (<200 nm in size), even in a heterogeneous mixture. MicroED data is acquired on a cryo-transmission electron microscope (cryo-TEM), using electrons as the incident beam.
Since MicroED is a diffraction technique, samples need to be crystalline, and the crystallization process is the same as for X-ray crystallography. However, much smaller crystals can be used in MicroED because the interaction of the crystal with electrons is much stronger than it is with X-rays. Crystals that are only 100 nm in size can readily be analyzed. This may significantly shorten the sample preparation process and allows for the analysis of crystals that are too small to diffract with other methods. Data collection is completed in only a few minutes, and 3D structures can be determined at atomic resolution.
Sample requirements for MicroED
The unique requirements of MicroED set it apart, not just from other cryo-electron microscopy (cryo-EM) techniques, but from traditional crystallography as well. MicroED necessitates small crystals (<50 µm) but samples can be cut (or milled) from larger crystals using a focused ion beam (FIB), if necessary. (Note that crystals >200 nm have increased secondary scattering, which convolutes the data.)
Sample preparation also varies between small molecule and protein samples. Small molecule crystals are usually dry and can often be analyzed at room temperature. Mechanical grinding can easily be used to reduce the size of large crystals, or the molecules can simply be crystallized spontaneously out of solution using evaporation.
Proteinase K structure. MicroED data provided by the Gonen Laboratory HHMI/UCLA.
Protein crystals, however, are typically kept in water to retain their hydrated, native states. These samples are subsequently flash-frozen (vitrified) in order to avoid sample damage due to crystalline ice formation. Vitrified samples are sensitive to changes in humidity or to the buffer and may disintegrate at the slightest touch. Therefore, cryo-FIB milling is used to reduce the size of large protein crystals.
What are they key benefits of MicroED?
MicroED fills a gap in structural biology, since X-ray crystallography requires large crystals that can be difficult to obtain whereas synchrotron X-ray free-electron lasers (XFELs) necessitates hundreds, if not thousands, of small crystals for the structural analysis of a single protein or small molecule. Time on a synchrotron is also expensive and access is limited. MicroED uniquely offers fast, high-resolution analysis that requires only a few small crystals.
Fast, atomic-resolution 3D structural information
Obtain diffraction data from nanocrystals in minutes.
Instant productivity
Nanocrystals as small as 100 nm can readily be analyzed, removing the burden of large crystal growth (as needed in X-ray crystallography). This also reduces the amount of sample material required. Mixtures of different polymorphs and compounds can be analyzed.
Complete turnkey solution
Obtain hardware, software, and support from one single vendor. Acquired data can be processed using established reconstruction packages for X-ray crystallography.
2-in-1 solution
MicroED and single particle analysis can be performed on the same cryo-electron microscope. This solution is compatible with new microscopes but can also be retrofitted onto existing cryo-EM instruments.
MicroED and drug discovery
MicroED is becoming increasingly popular in drug discovery as it can be used to determine the structure of protein-drug complexes and small molecules. Critically, MicroED alleviates the need for long and complicated large-crystal growth, a significant benefit in the fast-paced pharmaceutical industry.
To highlight some of the key benefits of MicroED, we determined the structure of acetaminophen from a commercial sample. Only ~10-12 grams of the sample were required to obtain the structure shown; the nanocrystal was also extracted from a mixture consisting of filler and other compound constituents. The entire 70-degree range of data was collected in a matter of minutes.
Example of MicroED structure determination performed in-house on Thermo Scientific instrumentation.
Data processing was performed in the open-source Diffraction Integration for Advanced Light Sources (DIALS) software, as outlined below. It is important to note that while the full structure could be determined from a dataset that was only 43% complete, additional data from other crystals could be used to further enhance these results.
General outline of MicroED data processing performed with open-source Diffraction Integration for Advanced Light Sources (DIALS) Software.
MicroED solutions
With the addition of our complete MicroED solution, your Thermo Scientific cryo-TEM becomes your own in-house beam line for structure determination!
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An example of a typical X-ray crystallography and MicroED pipeline.
A typical MicroED workflow.
The evolution of MicroED as told by key publications in the field. Articles are color-coded by sample type and preparation method.
Documents
Structural Biology Research
Cryo-electron microscopy enables the structural analysis of challenging biological targets such as large complexes, flexible species and membrane protein.
Drug Discovery
Learn how to take advantage of rational drug design for many major drug target classes, leading to best-in-class drugs.
Proteins Analysis
Cryo-electron microscopy provides near-atomic resolution 3D protein structure. It can determine structural information for complexes and crystallization-resistant samples, as well as vital cellular context.
Biopharmaceutical Research
Structural drug discovery is enabled by cryo-electron microscopy, as the method provides near-atomic-resolution detail for small molecules and protein biologics in their fully hydrated state.
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