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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. Click through the steps below for more details on each step in preparing a cryo-EM single particle sample suitable for high-resolution data collection:
Protein expression
Using the right protein expression system for cryo-EM is critical to success. Protein solubility, functionality, purification speed, and yield should be considered 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 for single particle Cryo-EM sample preparation
High-quality protein purification is essential for successful single particle cryo-EM, as sample quality directly impacts achievable resolution and data consistency.
Although single particle analysis can address some heterogeneity through 3D classification, well-defined and stable samples are critical for reliable vitrification and structural reconstruction. Affinity chromatography is commonly used for protein or antibody isolation, often followed by protease-based tag removal.
Buffer composition and additive selection play a key role in maintaining sample stability, and optimized conditions help minimize aggregation, dissociation, and preferred particle orientation on cryo-EM grids.
Screening cryo-EM–compatible buffers and detergents, such as those included with the Thermo Scientific VitroEase Buffer Screening Kit, supports identification of conditions that preserve protein integrity under cryogenic conditions. Detergents can also be used to improve protein extraction and stability across a range of sample types.
Methods to improve sample imaging performance
High-quality cryo-EM data depends on structurally stable and conformationally homogeneous samples. In addition to protein purification, targeted optimization strategies can improve sample integrity, particle behavior, and overall imaging performance.
Cross-linking: Used for fragile or dynamic macromolecular assemblies, particularly those stabilized by weak interfaces that may dissociate during purification, blotting, or vitrification. Controlled, mild cross-linking reduces subunit dissociation and conformational flexibility, improving particle integrity, 2D class averages, and the proportion of usable particles. Common approaches include GraFix and AgarFix.
For more information, visit Protein Crosslinking Resources
Selected References:
- Stark. GraFix: stabilization of fragile macromolecular complexes for single particle cryo-EM. Methods in Enzymol. 481: 109-26 (2010). Doi: 10.1016/S0076-6879(10)81005-5
- Adamus et al. AgarFix: Simple and accessible stabilization of challenging single-particle cryo-EM specimens through crosslinking in a matrix of agar. J Struct Biol 207:327-331 (2019). DOI: 10.1016/j.jsb.2019.07.004
Protein scaffolding: Used with small proteins (typically <50 kDa) to increase apparent molecular weight and improve particle visibility in cryo-EM. By binding or fusing the target protein to rigid scaffolds, such as designed protein cages, DARPins, nanobodies, or legobodies, this approach enhances signal-to-noise ratio, supports particle alignment, and enables higher-resolution structural analysis.
Selected References:
- Yeates, Agdanowski and Liu. Development of Imaging Scaffolds for Cryo-Electron Microscopy. Curr Opin Struct Biol 60: 142-149 (2021). Doi: 10.1016/j.sbi.2020.01.012
- Wentinck, Gogou and Meijer. Putting on molecular weight: Enabling cryo-EM structure determination of sub-100-kDa proteins. Curr Res Struct Biol 4: 332-337 (2022). DOI: 10.1016/j.crstbi.2022.09.005
Cryo-EM sample quality assessment
After purification, sample quality should be evaluated to determine suitability for cryo-EM analysis.
Biochemical techniques such as differential scanning fluorimetry, SEC-MALS, and mass photometry provide insight into sample stability, size, and monodispersity. However, these methods may not fully capture compositional or conformational heterogeneity.
Negative-stain electron microscopy offers a rapid way to assess particle integrity and homogeneity at the molecular level, enabling visualization of sample quality prior to vitrification. Screening can be performed quickly on a standard TEM using a small number of grids. Ready-to-use negative stains can help streamline this step.
Native mass spectrometry provides an additional approach to evaluate sample stability, aggregation, and heterogeneity. Rapid screening, often within minutes per sample, can be achieved using tools such as Thermo Scientific NativePac OBE Columns.
Cryo-EM vitrification for single particle analysis
Cryo-EM samples are rapidly cooled to preserve their native structure and maintain compatibility with the high-vacuum environment of the electron microscope.
This rapid cooling prevents the formation of crystalline ice and instead produces vitreous (amorphous) ice, preserving the structural integrity of the biological specimen.
During vitrification, the sample is embedded in a thin layer of vitreous ice, where particles are evenly distributed and immobilized in near-native conditions. Maintaining this state requires handling and imaging at liquid nitrogen temperatures.
Vitrification is typically performed using semi-automated plunge freezing systems, which provide control over parameters such as blotting and freezing conditions to improve reproducibility.
Standard samples, such as apoferritin, are commonly used for workflow optimization, training, and quality control in single particle analysis.
Cryo-EM grid screening
After vitrification, samples are evaluated through cryo-EM grid screening prior to high-resolution data collection. This step determines whether the sample is suitable for downstream analysis, including 2D classification and initial 3D reconstruction.
Key parameters assessed during screening include particle distribution, concentration, and stability, as well as ice thickness, quality, and uniformity across the grid.
If these criteria are met, larger datasets can be collected for further 2D and 3D analysis. At this stage, moderate-resolution reconstructions are typically sufficient to guide sample optimization.
Screening is commonly performed on dedicated cryo-TEM platforms such as the Thermo Scientific Glacios 3 Cryo-TEM and Thermo Scientific Tundra Cryo-TEM, which support rapid evaluation and transfer of samples to higher-resolution systems, such as the Thermo Scientific Krios 5 Cryo-TEM.
Automated loading with AutoGrid sample carriers supports consistent handling across the workflow. Software solutions such as Thermo Scientific Smart EPU Software enable higher-throughput screening and efficient data collection.
Single particle cryo-EM sample preparation resources
For Research Use Only. Not for use in diagnostic procedures.