High-throughput crystallization of membrane proteins using the lipidic bicelle method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent's inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization. Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. Bicelles have been successfully used to crystallize several membrane proteins (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer's arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.     

Membrane protein crystallization in meso: lipid type-tailoring of the cubic phase

Hydrated monoolein forms the cubic-Pn3m mesophase that has been used for in meso crystallization of membrane proteins. The crystals have subsequently provided high-resolution structures by crystallographic means. It is possible that the hosting cubic phase created by monoolein alone, which itself is not a common membrane component, will limit the range of membrane proteins crystallizable by the in meso method. With a view to expanding the range of applicability of the method, we investigated by x-ray diffraction the degree to which the reference cubic-Pn3m phase formed by hydrated monoolein could be modified by other lipid types. These included phosphatidylcholine (PC), phosphatidylethanolamine, phosphatidylserine, cardiolipin, lyso-PC, a polyethylene glycol-lipid, 2-monoolein, oleamide, and cholesterol. The results show that all nine lipids were accommodated in the cubic phase to some extent without altering phase identity. The positional isomer, 2-monoolein, was tolerated to the highest level. The least well tolerated were the anionic lipids, followed by lyso-PC. The others were accommodated to the extent of 20-25 mol %. Beyond a certain concentration limit, the lipid additives either triggered one or a series of phase transitions or saturated the phase and separated out as crystals, as seen with oleamide and cholesterol. The series of phases observed and their order of appearance were consistent with expectations in terms of interfacial curvature changes. The changes in phase type and microstructure have been rationalized on the basis of lipid molecular shape, interfacial curvature, and chain packing energy. The data should prove useful in the rational design of cubic phase crystallization matrices with different lipid profiles that match the needs of a greater range of membrane proteins.     

Membrane-protein stability in a phospholipid-based crystallization medium

Protein stability is a crucial factor to consider when attempting to crystallize integral membrane proteins. Cubic phase, or in meso, lipid-bilayer crystallization media are thought to provide native-like environments that should facilitate membrane protein crystallization by helping to stabilize the native protein conformation for the duration of the crystallization process. While excellent crystals of bacteriorhodopsin (bR) and other Halobacterial rhodopsins have been obtained in lipid-bilayer gels formed with monoglycerides, success remains elusive in the general application of such media to other membrane proteins. Additionally, we have noted that some mutants of bR are highly unstable in gels formed with monoolein. Phosphatidylethanolamines (PE) and derivatives of PE represent another class of lipids that can form connected-bilayer gels. When wildtype bR and a labile bR mutant were reconstituted into this phospholipid gel, spectroscopy showed that the protein is both more stable and has improved conformational homogeneity as compared to gels formed using monoolein. In addition, we demonstrate that well-diffracting crystals of bR can be grown from a PE-based crystallization medium. Since most proteins lack a stability-indicating chromophore and other structure-based analytical techniques are poorly compatible with the lipid gel, we developed a generally-applicable spectroscopic technique based on the intrinsic fluorescence of tryptophan residues. This fluorescence assay makes possible the rapid evaluation of lipid gels as media for the crystallization of membrane proteins.     

Lipidic cubic phase technologies for membrane protein structural studies

Lipidic cubic phase (LCP) is a membrane-mimetic matrix suitable for stabilization and crystallization of membrane proteins in lipidic environment. LCP technologies, however, have not been fully embraced by the membrane protein structural biology community, primarily because of the difficulties associated with handling viscous materials. Recent developments of pre-crystallization assays and improvements in crystal imaging, successes in obtaining high resolution structures of G protein-coupled receptors (GPCRs), and commercial availability of LCP tools and instruments are beginning to attract structural biologists to integrate LCP technologies in their research. This wider acceptance should translate to an increased number of otherwise difficult-to-crystallize membrane protein structures, shedding light on their functional mechanisms and on structural details of lipid-protein interactions.     

Effect of phospholipids and a transmembrane peptide on the stability of the cubic phase of monoolein: implication for protein crystallization from a cubic phase

The cubic phase of monoolein has successfully been used for crystallization of a number of membrane proteins. However, the mechanism of protein crystallization in the cubic phase is still unknown. It was hypothesized, that crystallization occurs at locally formed patches of bilayers. To get insight into the stability of the cubic phase, we investigated the effect of different phospholipids and a model transmembrane peptide on the lipid organization in mixed monoolein systems. Deuterium-labeled 1-oleoyl-rac-[(2)H(5)]-glycerol was used as a selective probe for (2)H NMR. The phase behavior of the phospholipids was followed by (31)P NMR. Upon incorporation of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, or phosphatidic acid, the cubic phase of monoolein transformed into the L(alpha) or H(II) phase depending on the phase preference of the phospholipid and its concentration. The ability of phospholipids to destabilize the cubic phase was found to be dependent on the phospholipid packing properties. Electrostatic repulsion facilitated the cubic-to-L(alpha) transition. Incorporation of the transmembrane peptide KALP31 induced formation of the L(alpha) phase with tightly packed lipid molecules. In all cases when phase separation occurs, monoolein and phospholipid participate in both phases. The implications of these findings for protein crystallization are discussed.     

Monoolein lipid phases as incorporation and enrichment materials for membrane protein crystallization

The crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established methodology in many structural biology laboratories. The standard procedure employed with this methodology requires the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial in-situ enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases the efficiency of identifying productive crystallization parameters. Finally, we provide a model that explains the incorporation of the membrane protein from solution into the lipid phase via a portal lamellar phase.     

An automated pipeline to screen membrane protein 2D crystallization

Electron crystallography relies on electron cryomicroscopy of two-dimensional (2D) crystals and is particularly well suited for studying the structure of membrane proteins in their native lipid bilayer environment. To obtain 2D crystals from purified membrane proteins, the detergent in a protein–lipid–detergent ternary mixture must be removed, generally by dialysis, under conditions favoring reconstitution into proteoliposomes and formation of well-ordered lattices. To identify these conditions a wide range of parameters such as pH, lipid composition, lipid-to-protein ratio, ionic strength and ligands must be screened in a procedure involving four steps: crystallization, specimen preparation for electron microscopy, image acquisition, and evaluation. Traditionally, these steps have been carried out manually and, as a result, the scope of 2D crystallization trials has been limited. We have therefore developed an automated pipeline to screen the formation of 2D crystals. We employed a 96-well dialysis block for reconstitution of the target protein over a wide range of conditions designed to promote crystallization. A 96-position magnetic platform and a liquid handling robot were used to prepare negatively stained specimens in parallel. Robotic grid insertion into the electron microscope and computerized image acquisition ensures rapid evaluation of the crystallization screen. To date, 38 2D crystallization screens have been conducted for 15 different membrane proteins, totaling over 3000 individual crystallization experiments. Three of these proteins have yielded diffracting 2D crystals. Our automated pipeline outperforms traditional 2D crystallization methods in terms of throughput and reproducibility.     

Two-dimensional crystallization of membrane proteins: the lipid layer strategy

Due to the difficulty to crystallize membrane proteins, there is a considerable interest to intensify research topics aimed at developing new methods of crystallization. In this context, the lipid layer crystallization at the air/water interface, used so far for soluble proteins, has been recently adapted successfully to produce two-dimensional (2D) crystals of membrane proteins, amenable to structural analysis by electron crystallography. Besides to represent a new alternative strategy, this approach gains the advantage to decrease significantly the amount of material needed in incubation trials, thus opening the field of crystallization to those membrane proteins difficult to surexpress and/or purify. The systematic studies that have been performed on different classes of membrane proteins are reviewed and the physico-chemical processes that lead to the production of 2D crystals are addressed. The different drawbacks, advantages and perspectives of this new strategy for providing structural information on membrane proteins are discussed.     

Role of glycosylation and membrane environment in nicotinic acetylcholine receptor stability

The effects of glycosylation and membrane environment on the structural stability of the nicotinic acetylcholine receptor (nAChR) from Torpedo have been investigated to improve our understanding of factors that influence eukaryotic membrane protein crystallization. Gel shift assays and carbohydrate-specific staining show that the deglycosylation enzyme, Endo F1, removes at least 50% of membrane-reconstituted nAChR glycosylation. The extent of deglycosylation with Endo F1 increases upon detergent solubilization. Removal of between 60-100% of high mannose moieties from the nAChR has no effect on nAChR secondary structure, stability, or flexibility. Deglycosylation does not influence either agonist binding or the ability of the nAChR to undergo agonist-induced conformational change. In contrast, nAChR structural stability, flexibility, and function are all negatively influenced by simple changes in reconstituted membrane lipid composition. Our results suggest that deglycosylation may represent a feasible approach for enhancing the crystallizability of the nAChR. Our data also demonstrate that the dependence of nAChR structural stability on lipid environment may represent a significant obstacle to nAChR crystallization. Some membrane proteins may have evolved complex interactions with their lipid environments. Understanding the complexity of these interactions may be essential for devising an appropriate strategy for the crystallization of some membrane proteins.     

Data mining crystallization databases: knowledge-based approaches to optimize protein crystal screens

Protein crystallization is a major bottleneck in protein X-ray crystallography, the workhorse of most structural proteomics projects. Because the principles that govern protein crystallization are too poorly understood to allow them to be used in a strongly predictive sense, the most common crystallization strategy entails screening a wide variety of solution conditions to identify the small subset that will support crystal nucleation and growth. We tested the hypothesis that more efficient crystallization strategies could be formulated by extracting useful patterns and correlations from the large data sets of crystallization trials created in structural proteomics projects. A database of crystallization conditions was constructed for 755 different proteins purified and crystallized under uniform conditions. Forty-five percent of the proteins formed crystals. Data mining identified the conditions that crystallize the most proteins, revealed that many conditions are highly correlated in their behavior, and showed that the crystallization success rate is markedly dependent on the organism from which proteins derive. Of the proteins that crystallized in a 48-condition experiment, 60% could be crystallized in as few as 6 conditions and 94% in 24 conditions. Consideration of the full range of information coming from crystal screening trials allows one to design screens that are maximally productive while consuming minimal resources, and also suggests further useful conditions for extending existing screens.     

Femtosecond crystallography of membrane proteins in the lipidic cubic phase

Despite recent technological advances in heterologous expression, stabilization and crystallization of membrane proteins (MPs), their structural studies remain difficult and require new transformative approaches. During the past two years, crystallization in lipidic cubic phase (LCP) has started gaining a widespread acceptance, owing to the spectacular success in high-resolution structure determination of G protein-coupled receptors (GPCRs) and to the introduction of commercial instrumentation, tools and protocols. The recent appearance of X-ray free-electron lasers (XFELs) has enabled structure determination from substantially smaller crystals than previously possible with minimal effects of radiation damage, offering new exciting opportunities in structural biology. The unique properties of LCP material have been exploited to develop special protocols and devices that have established a new method of serial femtosecond crystallography of MPs in LCP (LCP-SFX). In this method, microcrystals are generated in LCP and streamed continuously inside the same media across the intersection with a pulsed XFEL beam at a flow rate that can be adjusted to minimize sample consumption. Pioneering studies that yielded the first room temperature GPCR structures, using a few hundred micrograms of purified protein, validate the LCP-SFX approach and make it attractive for structure determination of difficult-to-crystallize MPs and their complexes with interacting partners. Together with the potential of femtosecond data acquisition to interrogate unstable intermediate functional states of MPs, LCP-SFX holds promise to advance our understanding of this biomedically important class of proteins.     

The effect of bacteriorhodopsin, detergent and hydration on the cubic-to-lamellar phase transition in the monoolein-distearoyl phosphatidyl glycerol-water system

The cubic phase of monoolein (MO) has successfully been used for crystallization of membrane proteins. It is likely that the transition to a lamellar phase upon dehydration is important for the crystallization process, and that the internal dimensions of the lipid phases (i.e., water pore diameter) are crucial for the inclusion and the diffusion of membrane proteins. In the present study, we investigated the cubic-to-lamellar phase transitions in the MO-water and the MO-distearoyl phosphatidyl glycerol (DSPG) systems. The MO-water system was investigated by means of isothermal sorption and desorption microcalorimetry. We show that the transition from cubic to lamellar phase induced by desorption is driven by entropy. At 25 degrees C, this occurs at a water activity of 0.98 with a transition enthalpy of 860 J/mol (MO). The phase behavior was also investigated in the presence of a small amount of the transmembrane protein bacteriorhodopsin (bR), and a detergent, octyl glucoside (OG), and it was shown that both bR and OG stabilize the lamellar phase. Analogous results were obtained for the MO-DSPG-water system. The latter system resembles the MO-water system in that a cubic-to-lamellar phase transition is induced by dehydration, although the structural properties of these phases are slightly different. Finally, we demonstrate that bR can be crystallized from a cubic phase of MO-DSPG-buffer.     

Crystallization screens: compatibility with the lipidic cubic phase for in meso crystallization of membrane proteins.

The in meso method for growing crystals of membrane proteins uses a spontaneously forming lipidic cubic mesophase. The detergent-solubilized protein is dispersed with lipid, typically monoolein, and in so doing the cubic phase self-assembles. A precipitant is added to trigger crystal nucleation and growth. The commercial screen solution series are convenient for use in crystallization trials. The aim of this study was to determine which of the Hampton Screen and Screen 2 series of solutions are compatible with the in meso method. These screens contain components any of which could destroy the cubic phase. X-ray diffraction was used for phase identification and for microstructure characterization. The study was done at 4 degrees C and at 20 degrees C. Two types of sample preparations were examined. One used an excess of half-strength screen solution (Prep. 1). The other used a limiting quantity of undiluted screen solution (Prep. 2). At 20 degrees C, over 90% of the screen solutions produced the cubic phase with Prep. 1. This figure dropped to 50% with Prep. 2. In contrast, 50 to 60% of the screens were cubic phase compatible at 4 degrees C under Prep. 1 conditions. The figure fell to 25% with Prep. 2. The mode of action of the diverse screen components are explained on the basis of the phase properties of the monoolein/water system.     

Membrane protein structure determination by electron crystallography

During the past year, electron crystallography of membrane proteins has provided structural insights into the mechanism of several different transporters and into their interactions with lipid molecules within the bilayer. From a technical perspective there have been important advances in high-throughput screening of crystallization trials and in automated imaging of membrane crystals with the electron microscope. There have also been key developments in software, and in molecular replacement and phase extension methods designed to facilitate the process of structure determination.     

Detergents destabilize the cubic phase of monoolein: implications for membrane protein crystallization

The in meso method for membrane protein crystallization uses a lipidic cubic phase as the hosting medium. The cubic phase provides a lipid bilayer into which the protein presumably reconstitutes and from which protein crystals nucleate and grow. The solutions used to spontaneously form the protein-enriched cubic phase often contain significant amounts of detergents that were employed initially to purify and to solubilize the membrane protein. By virtue of their surface activity, detergents have the potential to impact on the phase properties of the in meso system and, by extension, the outcome of the crystallization process. The purpose of this study was to quantify the effects that a popular series of nonionic detergents, the n-alkyl-beta-D-glucopyranosides, have on the phase behavior of hydrated monoolein, the lipid upon which the in meso method is based. Phase identity and phase microstructure were characterized by small-angle x-ray diffraction on samples prepared to mimic in meso crystallization conditions. Measurements were made in the 0-40 degrees C range. Samples prepared in the cooling direction allow for the expression of metastability, a feature of liquid crystalline phases that might be exploited in low-temperature crystallization. The results show that the cubic phase is relatively insensitive to small amounts of alkyl glucosides. However, at higher levels the detergents trigger a transition to the lamellar phase in a temperature- and salt concentration-dependent manner. These effects have important implications for in meso crystallization. A diffraction-based method for assaying detergents is presented.     

Use of octyl beta-thioglucopyranoside in two-dimensional crystallization of membrane proteins

A great interest exists in producing and/or improving two-dimensional (2D) crystals of membrane proteins amenable to structural analysis by electron crystallography. Here we report on the use of the detergent n-octyl beta-d-thioglucopyranoside in 2D crystallization trials of membrane proteins with radically different structures including FhuA from the outer membrane of Escherichia coli, light-harvesting complex II from Rubrivivax gelatinosus, and Photosystem I from cyanobacterium Synechococcus sp. We have analyzed by electron microscopy the structures reconstituted after detergent removal from lipid-detergent or lipid-protein-detergent micellar solutions containing either only n-octyl beta-d-thioglucopyranoside or n-octyl beta-d-thioglucopyranoside in combination with other detergents commonly used in membrane protein biochemistry. This allowed the definition of experimental conditions in which the use of n-octyl beta-d-thioglucopyranoside could induce a considerable increase in the size of reconstituted membrane structures, up to several micrometers. An other important feature was that, in addition to reconstitution of membrane proteins into large bilayered structures, this thioglycosylated detergent also was revealed to be efficient in crystallization trials, allowing the proteins to be analyzed in large coherent two-dimensional arrays. Thus, inclusion of n-octyl beta-d-thioglucopyranoside in 2D crystallization trials appears to be a promising method for the production of large and coherent 2D crystals that will be valuable for structural analysis by electron crystallography and atomic force microscopy.     

Determination and application of empirically derived detergent phase boundaries to effectively crystallize membrane proteins

Elucidating the structures of membrane proteins is essential to our understanding of disease states and a critical component in the rational design of drugs. Structural characterization of a membrane protein begins with its detergent solubilization from the lipid bilayer and its purification within a functionally stable protein‐detergent complex (PDC). Crystallization of the PDC typically occurs by changing the solution environment to decrease solubility and promote interactions between exposed hydrophilic surface residues. As membrane proteins have been observed to form crystals close to the phase separation boundaries of the detergent used to form the PDC, knowledge of these boundaries under different chemical conditions provides a foundation to rationally design crystallization screens. We have carried out dye‐based detergent phase partitioning studies using different combinations of 10 polyethylene glycols (PEG), 11 salts, and 11 detergents to generate a significant amount of chemically diverse phase boundary data. The resulting curves were used to guide the formulation of a 1536‐cocktail crystallization screen for membrane proteins. We are making both the experimentally derived phase boundary data and the 1536 membrane screen available through the high‐throughput crystallization facility located at the Hauptman‐Woodward Institute. The phase boundary data have been packaged into an interactive Excel spreadsheet that allows investigators to formulate grid screens near a given phase boundary for a particular detergent. The 1536 membrane screen has been applied to 12 membrane proteins of unknown structures supplied by the structural genomics and structural biology communities, with crystallization leads for 10/12 samples and verification of one crystal using X‐ray diffraction.     

High-resolution AFM of membrane proteins directly incorporated at high density in planar lipid bilayer

The heterologous expression and purification of membrane proteins represent major limitations for their functional and structural analysis. Here we describe a new method of incorporation of transmembrane proteins in planar lipid bilayer starting from 1 pmol of solubilized proteins. The principle relies on the direct incorporation of solubilized proteins into a preformed planar lipid bilayer destabilized by dodecyl-beta-maltoside or dodecyl-beta-thiomaltoside, two detergents widely used in membrane biochemistry. Successful incorporations are reported at 20 degrees C and at 4 degrees C with three bacterial photosynthetic multi-subunit membrane proteins. Height measurements by atomic force microscopy (AFM) of the extramembraneous domains protruding from the bilayer demonstrate that proteins are unidirectionally incorporated within the lipid bilayer through their more hydrophobic domains. Proteins are incorporated at high density into the bilayer and on incubation diffuse and segregate into protein close-packing areas. The high protein density allows high-resolution AFM topographs to be recorded and protein subunits organization delineated. This approach provides an alternative experimental platform to the classical methods of two-dimensional crystallization of membrane proteins for the structural analysis by AFM. Furthermore, the versatility and simplicity of the method are important intrinsic properties for the conception of biosensors and nanobiomaterials involving membrane proteins.     

A model-based approach for mining membrane protein crystallization trials

MOTIVATION: Membrane proteins are known to play crucial roles in various cellular functions. Information about their function can be derived from their structure, but knowledge of these proteins is limited, as their structures are difficult to obtain. Crystallization has proved to be an essential step in the determination of macromolecular structure. Unfortunately, the bottleneck is that the crystallization process is quite complex and extremely sensitive to experimental conditions, the selection of which is largely a matter of trial and error. Even under the best conditions, it can take a large amount of time, from weeks to years, to obtain diffraction-quality crystals. Other issues include the time and cost involved in taking multiple trials and the presence of very few positive samples in a wide and largely undetermined parameter space. Therefore, any help in directing scientists' attention to the hot spots in the conceptual crystallization space would lead to increased efficiency in crystallization trials. RESULTS: This work is an application case study on mining membrane protein crystallization trials to predict novel conditions that have a high likelihood of leading to crystallization. We use suitable supervised learning algorithms to model the data-space and predict a novel set of crystallization conditions. Our preliminary wet laboratory results are very encouraging and we believe this work shows great promise. We conclude with a view of the crystallization space that is based on our results, which should prove useful for future studies in this area.     

Membrane protein crystallization in lipidic mesophases: detergent effects

The "cubic phase method" for growing crystals of membrane proteins uses a complex mixture of water, lipid, protein, and other components. The current view is that the cubic phase is integral to the process. Thus additives from whatever source introduce the possibility of destabilizing the phase, thereby compromising the crystallization process. Detergents are used to solubilize membrane proteins and are likely to be ported into the cubic medium with the target protein. Depending on the identity and concentration of the detergent, the cubic phase, which itself is membranous, may be solubilized or destabilized in such a way as to render it unsuitable as a crystal growing system. The nonionic detergent n-dodecyl-beta-D-maltopyranoside is commonly used in membrane protein work. In this study, we evaluate its effect on the cubic mesophase of hydrated monoolein. X-ray diffraction was used for phase identification and mesophase microstructure characterization. The results show that while low levels of the detergent are tolerated, increasing concentrations trigger a cubic-to-lamellar phase transition in a temperature-dependent manner. This finding is rationalized in the context of complementary molecular shapes of the lipid and the detergent and has implications for the mechanism of crystallization in lipidic mesophases as discussed.