Single-particle analysis (SPA) is a technique used in electron microscopy (EM) to determine the three-dimensional structure of a macromolecule or macromolecular complex, such as a protein or a virus. SPA involves imaging individual particles in many different orientations, and then combining these images to reconstruct a 3D model of the structure.
Cryo-electron microscopy (cryo-EM) is a type of EM that is commonly used for SPA. Cryo-EM involves freezing the sample in a thin layer of vitreous ice, which preserves the native state of the sample and allows for imaging at cryogenic temperatures (-160 to -180°C).
The basic steps involved in cryo-EM and SPA are as follows:
- Sample preparation: The sample is purified and concentrated, and then applied to a grid. The grid is blotted to remove excess liquid and then plunged into liquid ethane or propane to flash-freeze the sample in a thin layer of vitreous ice.
- Imaging: The sample grid is transferred to an electron microscope, and the particles are imaged in many different orientations using a low-dose electron beam. The images are collected using a digital camera and can be processed in real-time to assess image quality and particle distribution.
- Particle picking: The individual particles are identified and extracted from the images using computer software. The particles are then classified into groups based on their similarity.
- 3D reconstruction: The 2D images of the particles are combined to create a 3D reconstruction of the structure. This is done using computer software that calculates the orientations and positions of the particles.
- Model building and refinement: The 3D reconstruction is used to generate an atomic model of the structure, which is refined through iterative cycles of model building and refinement.
Cryo-EM and SPA have revolutionized the field of structural biology, allowing for the determination of high-resolution structures of macromolecules and macromolecular complexes that were previously inaccessible by other techniques. The technique is used in many areas of research, including drug discovery, biochemistry, and biophysics.
