Main Content
Applications
Single particle analysis is generally used to determine the 3D structure of globular proteins and protein complexes at high resolution.
Method
Grids holding purified protein samples are plunge-frozen in liquid ethane and imaged. Single protein particles are oriented in various directions, allowing the 3D structure of the complex to be reconstructed by image processing methods. Filaments require a different approach for processing images; e.g. helical reconstruction.
Advantages
The protein particles are imaged in a close-to-native environment. Only a small amount of protein solution is required. Crystalization is not necessary. The entire 3-D space is sampled. High resolution (~3Å or better) is reachable for 'large' proteins (mass >100kDa).
Disadvantages
High resolution is not as easily reachable for small proteins (mass <100kDa) although this is improving rapidly. The resolution might also be limited for proteins with a high degree of variability and flexibility such as protein complexes.
Sample requirements
Pure, stable and homogeneous protein samples; concentration higher than 0.2 mg/ml is generally required. At this concentration, we need minimally ~ 50 microliters of the solution. We would like to see a protein gel, and require full information about the buffer as well as anything known about protein stability. Buffers should ideally be low in salt <150 mM, not contain any cryo-protectants or sugars such as glycerol or sucrose. Additives such as DMSO and detergents should be avoided if possible.