Two-Dimensional Crystallization of Membrane Proteins by Reconstitution Through Dialysis
Studies of membrane proteins by two-dimensional (2D) crystallization and electron crystallography have provided crucial information on the structure and function of a rapidly growing number of these intricate proteins within a close-to-native lipid bilayer. Here we provide protocols for planning and executing 2D crystallization trials by detergent removal through dialysis, including the preparation of phospholipids and the dialysis setup. General factors to be considered, such as the protein preparation, solubilizing detergent, lipid for reconstitution, and buffer conditions are discussed. Several 2D crystallization conditions are highlighted that have shown great promise to grow 2D crystals within a surprisingly short amount of time. Finally, conditions for optimizing order and size of 2D crystals are outlined.
Key wordsTwo-dimensional crystallization 2D crystals Membrane protein Dialysis Lipid Detergent Electron crystallography
We wish to thank our colleagues in the laboratory as well as in the cryo-EM community for helpful discussions over the years. We are particularly indebted to our collaborators for entrusting us with their valuable samples. Research in the Schmidt-Krey laboratory contributing to some of these observations was funded by NIH grant HL090630 and the Integrative Biosystems Institute.
- 5.Kühlbrandt W (2003) Two-dimensional crystallization of membrane proteins: a practical guide. In: Schägger H, Hunte C (eds) Membrane protein purification and crystallization: a practical approach. Academic Press, San Diego, pp 253–284Google Scholar
- 9.Ryan C, Stokes DL, Chen M, Zhang Z, Hardwicke PM (2004) Effect of orthophosphate, nucleotide analogues, ADP, and phosphorylation on the cytoplasmic domains of Ca(2+)-ATPase from scallop sarcoplasmic reticulum. J Biol Chem 13:5380–5386Google Scholar