Single Particle Analysis

Applications: To determine the 3D structure of proteins and protein complexes at high resolution. To determine the structure of filaments and their subunits 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

Advantages: The protein particles are imaged in a close-to-native environment. Only a small amount of protein is required. Crystalization is not required. The whole 3-D space is sampled. High resolution (up to ~3Å) is reachable for 'large' proteins (mass >100kDa).

Disadvantages: High resolution is not reachable for small proteins (mass <100kDa). The resolution might also be limited for proteins with a high degree of flexibility.

Sample requirements: Pure, stable and homogeneous protein samples; concentration higher than 0.2 mg/ml is required  At this concentration, we need ~ 50 microliters of the solution. We would like to see a gel, and require full information about the buffer as well as anything known about protein stability.