Table 1.

Summary of strategies used to crystallize membrane proteins at each stage of the pipeline.

Stage in Pipeline	Strategy	Rationale	Caveats/Comments
Molecular Biology and Protein Expression	Bacterial orthologs	Bacterial proteins express better in E. coli, can be stable in a range of conditions, are not glycosylated, and structures are likely very similar	Bacterial proteins can differ somewhat in structure, especially if modular domains are missing/present; function may differ enough from human homolog to limit drug discovery
	Stabilizing mutations	Less conformational heterogeneity will promote crystal growth	Non-native structure will be solved, flexibility that may be relevant to function will be masked
	Expression platform	Yield and stability can be vastly different for expression platforms, both of which are important parameters for future success in structure determination	Human proteins expressed in E. coli will lack glycosylation, which may be functionally relevant; other expression platforms may yield insufficient quantities of protein even for low volume crystallization screening
Membrane Protein Purification	Detergent selection for membrane solubilization	Yield is important for adequate crystallization trials, and maintaining function in detergent is also a good prognosticator	Many detergents exist and certain detergents have been used successfully for several membrane proteins, finding a suitable detergent for any protein of interest is unpredictable. In addition, a functional assay may not be available but proxy biophysical properties such as stability and circular dichroism spectra can be substituted
	Detergent selection/combination for crystallization	Optimized detergent for solubilization may not be appropriate for crystallization but detergents can be exchanged by immobilizing the protein on a column and eluting with a new buffer	Each detergent examined for crystallization must be rescreened to identify conditions
Membrane Protein Crystallization	Lipid phases	Crystallization in a membrane environment is likely to be more stabilizing and facilitate two-dimensional ordering of the membrane protein lattice	Some specialized equipment (commercially available, relatively inexpensive) is needed to reliably generate cubic phases
	Ligand or other additive	Less conformational heterogeneity and increase in stability facilitate crystallization	In many cases, a good ligand is unknown
	Covalent: gene for chaperone inserted into membrane protein construct	Exquisite control over placement, can replace a disordered loop with more compact three dimensional soluble protein	Limited knowledge of the membrane protein renders this process trial-and-error; limited number of successful different examples of proteins utilizing this approach
	Non-covalent: high affinity complex obtained by hybridoma or library screening (antibody, non-antibody platforms)	Highly crystallizable fragment provides lattice contacts, library screening methods offer medium to high-throughput	Hybridoma technology is expensive and low-throughput but newer methods are increasingly accessible and generalizable