Cryo electron microscopy (cryo-EM) has become a mainstream tool for determining the structures of macromolecular complexes at the atomic resolution. It has many advantages over other techniques such as X-ray crystallography and nuclear magnetic resonance (NMR). However, it also entails several challenges, a major one being preparation of an ideal sample. Recent studies have identified that DNA sensors and inflammasomes often assemble into filamentous oligomers, which poses a unique set of challenges in preparing ideal samples for high-resolution reconstruction using cryo-EM. This chapter will discuss how to overcome several major issues in cryo-EM sample preparation including construct design, screening using negative stain (ns) EM, and tips on working with filamentous proteins.
Keywords: Cryo-EM; Filaments; Fusion tags; Negative stain EM; Sample preparation.
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