The interactions leading to crystallization of the integral membrane protein bacteriorhodopsin solubilized in n-octyl-b-D-glucoside were investigated. Osmotic second virial coefficients (B 22 ) were measured by selfinteraction chromatography using a wide range of additives and precipitants, including polyethylene glycol (PEG) and heptane-1,2,3-triol (HT). In all cases, attractive protein-detergent complex (PDC) interactions were observed near the surfactant cloud point temperature, and there is a correlation between the surfactant cloud point temperatures and PDC B 22 values. Light scattering, isothermal titration calorimetry, and tensiometry reveal that although the underlying reasons for the patterns of interaction may be different for various combinations of precipitants and additives, surfactant phase behavior plays an important role in promoting crystallization. In most cases, solution conditions that led to crystallization fell within a similar range of slightly negative B 22 values, suggesting that weakly attractive interactions are important as they are for soluble proteins. However, the sensitivity of the cloud point temperatures and resultant coexistence curves varied significantly as a function of precipitant type, which suggests that different types of forces are involved in driving phase separation depending on the precipitant used.Keywords: protein-detergent complex; membrane protein crystallization; cloud point temperature; osmotic second virial coefficient; self-interaction chromatography Membrane proteins represent one of the most significant challenges in modern structural biology. Genome sequence analysis indicates that 20%-40% of the open-reading frames of most organisms encode membrane proteins, yet they represent <1% of the available structures in the Protein Data Bank (PDB) (Wallin and von Heijne 1998;Wiener 2004). Although several structural genomics initiatives have recently begun to focus on membrane proteins-particularly G-protein coupled receptors (GPCRs)-difficulties in expression, purification, and crystallization remain (Loll 2003;Lundstrom 2004). In particular, identifying a suitable detergent to solubilize a given membrane protein, thereby forming a protein-detergent complex (PDC), and combining this with the appropriate additives and precipitants necessary for crystal formation while retaining PDC stability adds several variables that must be considered beyond those for soluble proteins (Rosenbusch 1990;Garavito et al. 1996). For example, including 24 common detergents and additives as variables in a typical crystallization screen using conventional 24-well formats can result in the need to sample >3000 potential solution conditions, which makes such efforts largely impractical for all but highly automated Abbreviations: C 8 bG 1 , n-octyl-b-D-glucoside; BR, bacteriorhodopsin; HT, heptane-1,2,3-triol; 1,6-HD, hexane-1,6-diol; 1,2-HD, hexane-1,2-diol; PEG, polyethylene glycol; SIC, self-interaction chromatography.Article published online ahead of print. Article and publi...