b'Cryo-EM techniques Protein sample preparation for Cryo-EM cools samples to cryogenic temperatures so quicklycryo-EM analysisthat it prevents water molecules from crystalizing, preserving theThe most commonly used cryo-EM method today is SPA, native sample structure. Once frozen, a range of EM techniquesin which purified proteins are imaged to high-resolution 3D can be used to visualize the specimen in 3D at a variety ofstructures. SPA relies on near instantaneous freezing of proteins resolutionsincluding near-atomic resolutionallowing forfrom solution to a thin layer of noncrystalline ice on a grid in a deeper, more comprehensive insights than previously possible. process called vitrification. This process allows for capturing the SPA protein in its native state and providing a protective environment Single particle analysis (SPA) enables near-atomic structuralfor subsequent electron-based imaging at high vacuum in the determination of challenging proteins and protein complexestransmission electron microscope. without the need for crystallization. In SPA, purified proteinsSample preparation is currently a major bottleneck in SPA. or protein complexes are suspended in amorphous (vitreous)Even though semiautomated vitrification robots like the Thermo ice through rapid plunge freezing, which preserves the nativeScientific Vitrobot Mark IV System help to make parts of structures of the samples. Transmission electron microscopythe grid preparation more robust, the process can still prove (TEM) is then used to collect numerous 2D snapshots of thechallenging as many different parameters contribute to the samples. As the proteins are oriented randomly within the ice,quality of the prepared grid. When beginning cryo-EM sample these images show the sample at various angles, and canpreparation, it is critical to have a well-characterized and be recombined into a high-resolution 3D reconstruction ofhomogeneous sample, preferably tested by negative stain the sample. imaging or mass spectrometry. Nevertheless, even purified MicroED proteins that are stable and structurally intact can behave Microcrystal electron diffraction (MicroED) enables fast,differently in a thin vitreous ice layer, exhibiting unwanted behavior high-resolution, structural determination of small moleculessuch as denaturation, aggregation, or preferred orientation. and proteins. Atomic details can be extracted from individualDue to the unique properties of each protein, multiple rounds of nanocrystals (200 nm in size), even in a heterogeneous mixture.optimization are often necessary, where vitrification parameters, Data is acquired by cryo-TEM, using electrons as the incidentgrid types, or additives are adjusted before the optimal condition beam. Since MicroED is a diffraction technique, samples need tofor high-resolution data collection are found (i.e., particles are be crystallized using the same methods that are found in X-raystructurally intact, randomly oriented, and equally distributed). crystallography. However, much smaller crystals (~100 nm in size)Currently, sample optimization is often performed in a can be used in MicroED because the interaction of the crystalnonsystematic way, extending optimization time over multiple with electrons is much stronger than its interaction with X-rays.days or even weeks.This may significantly shorten the sample preparation processIn order to support the successful preparation of cryo-EM and allow for the analysis of crystals that are too small to diffractsamples, we offer solutions for every step of the cryo-EM with other methods. workflow to help to train new users, optimize sample conditions, Cryo-ET and provide standards that can be used for benchmarking or workflow optimization (Figure 1). Cryo-electron tomography (cryo-ET) provides label-free, fixation-free, nanometer-scale imaging of a cells interior in 3D and visualizes protein complexes within their physiological environments. Using a correlative light and electron microscopy approach allows targeting of tagged proteins by fluorescence microscopy before subsequent cryo-EM higher-resolution imaging. Many cells are too thick for electrons, so the vitrified cells must be thinned with a cryo-focused ion beam (cryo-FIB) microscope prior to imaging in a transmission electron microscope.Cryo-EM sample preparation thermofisher.com/proteinbiology 113'