To determine the structure of filaments and their subunits at high resolution.
Grids holding purified filament samples are plunge-frozen in liquid ethane and imaged. The 3D structure(s) of helical assemblies is reconstructed from their diffraction patterns by image processing methods (helical analysis).
The protein particles are imaged in a close-to-native environment. If the filaments are helical, their subunits are present in a range of orientations.
The protein particles are imaged in a close-to-native 'frozen-hydrated' state. Only a small amount of protein is required. The whole 3-D space is automatically sampled if the filaments are helical. High resolution (to ~3Å) is reachable for well-ordered filamentous samples.
The resolution might be limited for filaments with a high degree of flexibility.
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.