Main Content
Protein Filaments
Applications
To determine the structure of filaments and their subunits at high resolution.
Method
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).
Advantages
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.
Advantages
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.
Disadvantages
The resolution might be limited for filaments 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.