Cryo-EM Sample Preparation

Protein preparation for structural biology

Any high-resolution cryo-EM structure starts with good protein preparation. Check out our Protein Sample Preparation eLearning Course to find out more about high-quality preparation of cryo-EM protein samples. 

Interested in further optimizing your protein production, purification, or biochemistry? Our sample preparation methods page contains a wide range of protein analysis techniques organized by sample type and method.  

After your sample is purified, it is important to assess whether the purification quality is suitable for further electron-microscopy analysis. Standard biochemical methods are not entirely sufficient, as solutions of intact complexes can actually consist of mixtures of compositionally different sub-complexes or structurally different conformations.

Negative-stain transmission electron microscopy

Negative-stain electron microscopy is an easy and straightforward method to assess the quality of purified biological specimens at the microscopic scale. The objective of this screening is to qualitatively assess particle composition and conformational homogeneity, which can only be done at the microscopic scale. Prepare negative stained samples more easily with ready-to-use VitroEase Methylamine Vanadate Negative Stain and VitroEase Methylamine Tungstate Negative Stain. 

Often this assessment is done on a simple side-entry microscope (e.g., a Thermo Scientific Talos L120C TEM), since screening is usually done one grid at a time, and the actual time spent on the microscope is short.

Cryo-EM sample conditions

Cryo-EM specimens are typically prepared using several microliters of protein solution at a concentration of 50 nM to 5 μM depending on the specimen, EM grid type, and other conditions used.

Single-particle analysis with cryo-EM depends on the computational averaging of thousands of images of identical particles, and therefore structural heterogeneity should be minimized in order to simplify structure determination.

Although the single particle analysis workflow can alleviate partial heterogeneity in the specimen with 3D classification procedures, biochemical purification of the sample (to isolate the target proteins) is required.

Apart from sample heterogeneity, it is also important that the particles on the cryo-EM grid are randomly orientated, so that views from all sides are available for reconstruction. Especially with asymmetric structures, preferred orientation can be problematic and significantly hinder the resolution of your structure. In these cases, adding in low concentrations of cryo-EM compatible detergents can help overcome this problem. Furthermore, detergents can help protect the proteins during the vitrification process from the air-water interface, which might denature unstable proteins so that they are no longer in their native state.

The VitroEase Buffer Screening Kit makes optimizing your cryo-EM samples easy by providing a selection of cryo-EM-compatible buffers and detergents that can be spiked directly into your samples for minimal sample manipulation and dilution.

EM sample preparation

For compatibility with the electron microscope vacuum, and to lock the individual particles in their native states, the solution containing the sample material must be frozen. In order to preserve the macromolecular structures, freezing has to happen rapidly enough to avoid crystalline ice formation; during vitrification an amorphous solid forms instead that does little or no damage to the sample structure. Afterwards, the sample must be kept at liquid nitrogen temperatures at all times to preserve the amorphous nature of the embedding ice layer and to avoid damage to the biological particles.

Vitrification

This operation produces a frozen hydrated sample, where the individual molecules of the specimen are well distributed and embedded in a very thin layer of amorphous (vitreous) ice.

The whole procedure can be simplified using semi-automated plungers such as the Thermo Scientific Vitrobot System and can be combined with the VitroEase Cryo-EM Training Kit, which can help users learn the intricacies of the vitrification process thanks to its detailed visual manual.

Sample quality in cryo-EM

Once the sample is vitrified, it should be evaluated with diagnostic cryo-EM before high-resolution data acquisition begins. The objective of this step is to qualitatively assess if the sample is a promising target for 2D class average analysis and, simultaneously, to obtain an initial low-resolution map. During this step the sample is pre-screened to evaluate the following properties:

• Protein concentration, stability, and distribution
• Ice quality, thickness, and uniformity across the grid

Cryo-electron microscopy screening

This screening ideally occurs on an Autoloader-based cryo-TEM system, as multiple freezing conditions can be screened without compromising ice quality. The Thermo Scientific Glacios Cryo-TEM, and Thermo Scientific Tundra Cryo-TEM are best suited for this purpose.

If the sample is promising, a larger set of images may be acquired to facilitate further 2D and 3D analyses. At this stage only a moderate resolution 3D map (> 3Å) is required.

Additionally, it has been shown that a 200 kV cryo-TEM equipped with direct detector cameras can produce high-resolution (<3 Å) data.

Glacios and Tundra Cryo-TEMs microscopes are ideally suited for this data acquisition and offer a robust and contamination-free designed-in connectivity with the higher resolution Krios Cryo-TEM. This allows for the exchange of AutoGrid cassettes and capsules between all Autoloader-equipped instruments.