Cryo-electron microscopy is a multi-step process – see how it can speed up your research and development by clicking the following workflow steps:
After your sample is purified it is important to assess whether the purification quality is suitable for further EM analysis. Standard biochemical methods are not entirely sufficient, as even intact complexes can be a mixture of compositionally different sub-complexes, and even homogeneous complexes can potentially adopt many 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.
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.
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.
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 grids, and other conditions used.
The biological specimen should remain active in the in vivo optimized conditions (buffer composition, etc.) for structural studies. A suitable biochemical or functional assay might also be exploited to test the activity of the protein.
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. A set of key parameters, such as specimen blotting time, blotting force, relative humidity and temperature, allows for reproducible preparation of high-quality vitrified specimens.
Samples 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
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 Talos Arctica 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.
The Glacios and Talos Arctica microscopes are ideally suited for this data acquisition and offer a robust and contamination-free designed-in connectivity with the higher resolution Thermo Scientific Krios cryo-TEM. This allows for the exchange of AutoGrid cassettes and capsules between all Autoloader equipped instruments.
The Thermo Scientific Krios G4 Cryo Transmission Electron Microscope (Cryo-TEM) is a highly stable 300 kV TEM platform complete with the industry-leading Autoloader, a cryogenic sample manipulation robot, making it the instrument of choice for high-resolution cryo-EM. Its long, unattended data collection results in unprecedented sample throughput and large-scale data acquisition capacity. With the Krios G4 Cryo-TEM you have the capability to obtain your structure at the highest possible resolution.
Once suitable cryo-EM grids have been identified with screening, a large dataset is collected to provide the highest possible resolution map. The Krios Cryo-TEM’s designed-in connectivity ensures a robust and risk-free pathway throughout the entire workflow, from sample preparation and optimization to image acquisition and data processing.
Centralized service facilities are a convenient option to receive access to cutting-edge cryo-EM technology and expertise.
The 3D reconstruction of macromolecules relies on the averaging of tens of thousands of particle views. For successful and efficient reconstruction, the data has to go through multiple steps of data processing, which is computationally expensive and requires that appropriate computational resources are available.
Careful consideration must therefore go into designing the data storage infrastructure to support Cryo-EM facilities, where terabytes of data are generated during data collection. Additionally, this large amount of data needs to be processed, and therefore computational resources are critical. A number of academic software packages have been developed specifically to aid in data processing.
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