Single Particle Analysis (SPA) is an electron microscopy method used to determine structures of proteins and other macromolecular complexes. Structures of macromolecules can be determined from cryo-EM images at near-atomic and sometimes true atomic resolution, and are used to investigate biological processes. It is an invaluable tool in multiple scientific fields, and although easy to explain, it is a complex process. This blog post will explain Single Particle Analysis and how it works.
What is Single Particle Analysis?
Single Particle Analysis (SPA) is a technique in structural biology used to determine the three-dimensional structure of proteins and other macromolecular complexes through the use of transmission electron microscopes (TEM). The technique involves the acquisition of high resolution images containing purified and monodisperse macromolecules and computationally extracting the individual particles from the images to reconstruct a final three-dimensional structure. The aim of SPA is to provide deep insights into structure and function of macromolecules, to help understand biological processes at the molecular level, and to help lead drug discovery efforts by providing details of molecular interactions. Therefore, SPA is performed under cryogenic conditions (cryo-EM), where macromolecular complexes are flash-frozen in liquid ethane so as to preserve their native state and kept at low temperature in the electron microscope during data collection. Post-acquisition computer processing involves complex alignments and classification of 2D particle images, which after merging can produce high-resolution 3D density maps , and provide atomic details of macromolecules. Alternatively, SPA can be performed under room temperature conditions on samples treated with negative stain producing lower resolution 3D structures, but which can still provide valuable information such as the shape and size of a macromolecule.
How Does Single Particle Analysis Work?
The working principle of SPA involves several physical and computational techniques that vary depending on the sample and desired outcome of the research. It is typically a complex process due to several factors such as the purity, stability, and heterogeneity of the macromolecular complex under study. The main advantage of cryo-EM and SPA is that it does not require large quantities of a sample. Yet, obtaining a 3D structure is time-consuming and involves processing large quantities of data. Therefore, the four main stages of SPA should be managed efficiently to ensure a successful experiment.
- Sample Preparation:
In order to obtain high-resolution structures through cryo-EM, samples must first undergo two fundamental processes: purification and vitrification. Samples are first isolated from biological sources and then further purified to provide highly pure and homogeneous samples in solution. Then the purified samples are frozen in a thin layer of vitreous ice, ready to be imaged in the cryo-electron microscope.
Before data collection, samples are first screened in a cryo-electron microscope to assess qualities such as protein concentration, stability, particle monodispersity and ice thickness. Then, once a suitable sample is identified, thousands of high-resolution images are collected with the help of automatic data collection software and used for further analysis.
- Image Processing:
Advanced image-processing software is used to process the images obtained through cryo-EM. The two-dimensional (2D) images are typically first automatically assessed for qualities such as resolution, motion, and ice thickness. Then, 2D images of individual particles are extracted and sorted into 2D classes based on similarity. Finally, these 2D classes are combined to reconstruct a final 3D density map.
- Analyzing and Interpreting Data:
Once a 3D density map has been produced, researchers fit the map with the appropriate amino acid chains and build a protein model guided by the map. Higher resolution 3D maps allow researchers to build models with high accuracy and confidence. The completed protein structures and corresponding density maps are then subjected to an analysis to elucidate the function of the complex under study. Researchers can then deposit their data in databases available to the public such as the Protein Data Bank (PDB) or the EMDB (Electron Microscopy Data Bank).
Applications of Single Particle Analysis
Single Particle Analysis is an invaluable tool in many scientific fields. Through the use of Transmission Electron Microscopes, researchers can better understand macromolecules such as membrane proteins, protein complexes, protein-DNA complexes, and nucleic acid structures. The sectors in which Single Particle Analysis is most commonly used include basic, biomedical, and pharmaceutical research.
JEOL and Single Particle Analysis
JEOL is a world leader in supplying electron microscopes, mass spectrometers, NMR spectrometers, and other high-end scientific equipment. Our mission is to offer advanced scientific and metrology products and services to support the development of a sustainable society.
JEOL offers two CRYO ARM™ models - 200kV and 300kV - that achieve unprecedented resolution and stability thus allowing for the automatic and unattended acquisition of image data for Single Particle Analysis. To learn more about what JEOL offers, please contact a member of the team today.
- 1. Dang, SY., and X., YX. (2022) Recent Technical Advances in Sample Preparation for Single-Particle Cryo-EM. Frontiers in Molecular Biosciences. Available from: https://www.frontiersin.org/articles/10.3389/fmolb.2022.892459
- 2. Sundaram, J. (20218) Single Particle Analysis Techniques. News Medical https://www.news-medical.net/life-sciences/Single-Particle-Analysis-Techniques.aspx
- 3. Weissenberger, G., Henderikx, R. J. M., and Peters, P. J. (2021). Understanding the Invisible Hands of Sample Preparation for Cryo-EM. Nat. Methods 18, 463–471. doi:10.1038/s41592-021-01130-6. Available from: https://scholar.google.com/scholar?hl=en&as_sdt=0%2C5&q=Understanding+the+Invisible+Hands+of+Sample+Preparation+for+Cryo-EM&btnG=