Search Thermo Fisher Scientific
- Contact Us
- Quick Order
-
Don't have an account ? Create Account
Single particle cryo-EM sample preparation workflow
A sample for single particle analysis is typically a vitrified suspension of biological material consisting of proteins, protein complexes, viruses or other macromolecules. As sample preparation is a vital step in the single particle workflow, it is important to have a good understanding of the steps involved and to have the right reagents, tools, and instrumentation. To prepare a cryo-EM single particle sample suitable for high-resolution data collection, the following steps are typically followed:
- Expression
- Purification
- Assessment
- Vitrification
- Screening
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
Although the single particle analysis workflow can resolve partial heterogeneity within the sample via 3D classification procedures, it is necessary to biochemically purify the sample containing the isolated target proteins. 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.
It is important to realize that the quality of the sample not only can contribute to a higher resolution, but that it can also affect the vitrification process itself. Optimization of the buffer and/or additive conditions can prevent aggregation, dissociation, and even preferred orientation of the particles on your grid during vitrification. To optimize your cryo-EM samples, consider using the Thermo Scientific VitroEase Buffer Screening Kit, which contains a broad selection of premade cryo-EM compatible buffers and detergents that is optimal for the samples’ stability in cryogenic conditions. We also offer a wide selection of detergents available in liquid and solid formats in flexible packaging to support protein extraction from a wide range of sample sources.
Cryo-EM sample quality assessment
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 consist of mixtures of compositionally different sub-complexes or structurally different conformations.
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. Negative stained samples can be prepared with ready-to-use Thermo Scientific VitroEase Methylamine Vanadate Negative Stain or Thermo Scientific VitroEase Methylamine Tungstate Negative Stain.
Often this assessment can be completed on a simple side-entry microscope (e.g., Thermo Scientific Talos L120C TEM), since screening is usually performed one grid at a time, and the actual time spent on the microscope is short.
Alternatively, Native Mass Spectrometry can be used to rapidly screen cryo-EM samples for biochemical stability, sample aggregation and conformational heterogeneity. Native mass spectrometry experiments can be done in as little as 3 minutes per sample using our nativePac-1 OBE columns.
Cryo-EM vitrification
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. 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 Thermo Scientific VitroEase Cryo-EM Training Kit, which can help users learn the intricacies of the vitrification process thanks to its detailed visual manual.
We also offer Thermo Scientific VitroEase Apoferritin Standard, which is a highly validated pure protein for use as a quality control sample for verifying microscope status, or for cryo-electron microscopy single particle analysis method development.
Cryo-EM grid screening
Once the sample is vitrified, it needs to 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
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.
Thermo Scientific Glacios 2 and Thermo Scientific Tundra Cryo-TEMs microscopes are ideally suited for this screening and offer a robust and contamination-free designed-in connectivity with higher resolution Thermo Scientific Krios Cryo-TEM. Glacios 2 Cryo-TEM allows for the exchange of cassettes and capsules between all Autoloader-equipped instruments and allows screening 12 samples at once with the optional Thermo Scientific EPU Multigrid Software. Tundra Cryo-TEM can visualize the impact of biochemical adjustments to samples by exchanging the samples in and out of the microscope in minutes, with nearly instantaneous feedback, allowing the optimization of the sample conditions quickly. The Tundra and Glacios 2 Cryo-TEMs use AutoGrid sample carriers, which are the industry standard for robust and reliable loading and unloading of cryogenic samples using a robotic sample loader. AutoGrids also enable the seamless interchange of samples between different microscopes in the workflow, so that sample preparation can be performed in a standard biochemistry laboratory.
Single particle cryo-EM sample preparation resources
For Research Use Only. Not for use in diagnostic procedures.