How Does MicroED Tackle Tricky Compounds That Don't Crystallize Well for X-ray?

For decades, X-ray crystallography has been the go-to method for determining the atomic structures of molecules. However, this powerful technique relies heavily on the ability to form well-ordered, large crystals, which can be a significant hurdle. Many important biological molecules, such as membrane proteins and large protein complexes, are notoriously difficult to crystallize. This is where MicroED, a rapidly advancing technique, steps in as a game-changer, often in conjunction with cryo-EM nature approaches.

The Crystallization Challenge

X-ray crystallography requires molecules to be arranged in a highly repetitive, three-dimensional lattice within a crystal. If the molecules are flexible, heterogeneous, or interact poorly with one another, forming crystals can be an immense challenge. This limitation has long hampered structural studies of many critical molecules, including those relevant to drug discovery and disease mechanisms.

MicroED: A Solution for Non-Crystallizable Compounds

MicroED (Microcrystal Electron Diffraction) offers a way to bypass the need for large, perfect crystals. Instead, it utilizes very small, sometimes even nano-sized, crystals or microcrystalline samples. Here’s how it works:

• Tiny Crystals, Big Impact: MicroED can analyze crystals that are far too small for traditional X-ray methods. These microcrystals can be formed from compounds that are extremely difficult to crystallize using conventional techniques.
• Electron Diffraction: Instead of X-rays, MicroED uses a beam of electrons to interact with the sample. When the electron beam passes through the microcrystal, it diffracts, creating a pattern that can be analyzed to determine the structure.
• Cryo-EM Nature: Often, MicroED is performed under cryogenic conditions (cryo-EM), which helps preserve the sample and reduce radiation damage. This combination, often called cryo-EM nature, enhances the quality of the data and enables the study of sensitive biological molecules.
• Overcoming the Size Barrier: MicroED overcomes the limitation of crystal size, making it possible to study molecules that were previously inaccessible through structural biology methods.

Key Advantages of MicroED:

• Versatility: MicroED is not limited by the type of molecule. It can handle proteins, peptides, small molecules, and even some inorganic compounds that are challenging to crystallize.
• Minimal Sample Requirements: Since it works with very small crystals, MicroED requires only minute amounts of sample. This is especially beneficial when dealing with scarce or difficult-to-produce biological samples.
• High Resolution: MicroED can achieve atomic-level resolution, providing detailed information about the structure of molecules.
• Complementary Technique: MicroED is a fantastic partner to techniques like cryo-EM nature based single-particle analysis (SPA), which studies individual molecules, and can confirm or complement results.

MicroED in Action: Real-World Examples

Several real world examples show the impact of microED, including:

• Drug Discovery: MicroED has been instrumental in determining the structures of drug candidates, especially those with complex chemistries or those that form microcrystals.
• Membrane Proteins: Membrane proteins, critical for cell function and drug targeting, are often notoriously difficult to crystallize. MicroED has allowed researchers to study these structures, enhancing our understanding of their mechanisms.
• Large Protein Complexes: MicroED has also contributed to structural studies of large protein complexes, which are often difficult to handle with traditional methods. In a recent study, cryo-EM with MicroED was utilized to reveal a supercomplex of plant plastid-encoded RNA polymerase, deciphering its structure and function.