January 1, 1970 (Vol. , No. )
Peter Nollert, Ph.D. Emerald Bio
Trying to grow membrane protein crystals? Read this first.
Protein crystals are necessary prerequisites for their detailed structural study by X-ray crystallography. Growing membrane protein crystals is typically a laborious trial and error-type search process that requires patience and willingness to explore alternatives outside of standard protocols. Here are eight often-overlooked tips to consider when embarking on a membrane protein crystallization project:
- Continue refining your membrane protein purification and sample preparation protocol. Good crystallizable membrane protein samples are often stable for extended periods of time, show a symmetric elution profile in a gel permeation chromatogram, and can be concentrated to fairly high concentrations often exceeding 20 mg/mL.
- Control the detergent concentration in the membrane protein sample. For many detergents this can be done by choosing the largest feasible MWCO (molecular weight cut off) filters during the concentration step or by dialysis. If all crystallization drops contain precipitate immediately after setup, the detergent concentration may be too low and too close to the detergent’s CMC (critical micellar concentration); therefore increasing its concentration is advised. Typically, though, the detergent concentration in the membrane protein sample is too high.
- Set up more crystallization experiments than you typically would for a soluble protein. It is important to capture a wide variety in crystallization parameters in the initial crystallization trials. Ten 96-well trays are a good starting point.
- Favor PEG-rich (polyethylene glycol) sparse matrix crystallization screens for initial membrane protein crystallization trials.
- Use the smallest volume your liquid dispensing tools can possibly work with. This conserves precious membrane protein sample.
- Take your time inspecting the crystallization drops by eye using a high-quality microscope. Search for membrane protein crystals that may be hidden behind precipitate, crystals that have no clear facets, or very small crystals. Switch between standard and highest possible optical magnification, and adjust the illumination angle if possible.
- Read Michael C. Wiener’s “A Pedestrian Guide to Membrane Protein Crystallization” for help in getting started or for troubleshooting.
- Explore nontraditional membrane protein crystallization regimes for the optimization of membrane protein crystal quality. For example, utilize bicelles, lipidic cubic phases, or just spiking of the membrane protein sample with lipids or amphiphilic reagents prior to subjecting it to a crystallization trial.
Peter Nollert, Ph.D., is Chief Technology Officer at Emerald Bio. To learn more about Emerald Bio services and products please visit Emerald Bio’s website.