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Microcrystal electron diffraction sample preparation workflow
Microcrystal electron diffraction (MicroED) enables fast, high-resolution, structural determination of small molecules and proteins. Atomic details can be extracted from individual nanocrystals (<200 nm in depth), even in a heterogeneous mixture. Data is acquired by cryo-TEM, using electrons as the incident beam. Samples must be crystallized for MicroED in order to provide the repeating lattice of molecules required to produce a lattice diffraction pattern. Sample preparation for MicroED varies between small molecule and protein samples, but similar methods to those used for X-ray crystallography can by used in both cases.
Protein sample preparation for MicroED
Protein expression
Using the right protein expression system for cryo-EM is critical to success. Consider protein solubility, functionality, purification speed, and yield when choosing an expression system. We offer a wide selection of protein expression systems to suit your research needs. Check out our protein expression system tool to find the right products for your research work.
Protein purification and optimization
It is critical to purify the protein in order to obtain a solution containing the isolated target proteins. The protein sample must be highly purified to crystalize. From a variety of reliable affinity chromatography reagents enabling protein and antibody purification, to an assortment of proteases for efficient tag removal, we have your needs covered. Check our protein sample preparation product options.
Sample vitrification
Microcrystals can be grown using standard methods, including vapour diffusion by hanging or sitting drop, under oil or batch crystallization. Conditions may have to be adapted, for example utilizing seeding, increased precipitant or increased protein concentration to encourage more nucleation, rather then the growth of a small number of large crystals, as is more commonly preferred. Once a shower of microcrystals has been produced, they can be deposited on EM grids by pipette, as with other protein samples. For vitrification, grids are blotted and plunged into liquid ethane using a Thermo Scientific Vitrobot System.
Cryo-FIB milling
If the crystals are too thick to be examined by cryo-transmission electron microscopy (cryo-TEM), the strong scattering of the electrons make it impossible to efficiently penetrate the crystals with the electron beam. Cryo-FIB milling can be used for thinning the microcrystals to thicknesses that are suitable for MicroED investigation.
Small molecule preparation for MicroED
Small molecule crystals are usually dry and can often be prepared at room temperature. Mechanical grinding can easily be used to reduce the size of large crystals, or crystallization conditions can be optimized to produce a shower of microcrystals via evaporation or other techniques. MicroED necessitates thin crystals (<200 nm) but samples can be cut or milled from larger crystals using a focused ion beam (FIB), if necessary. It's important to note that crystals thicker than 200 nm have increased secondary scattering, which convolutes the data.
Microcrystal electron diffraction sample preparation resources
For Research Use Only. Not for use in diagnostic procedures.