- 1. Introduction
- 2. A working model for in meso crystallization
- 3. The in meso method: practical issues and challenges
- 4. High-throughput crystallogenesis and the in meso robot
- 5. Mesophase compatibility with protein-solution components
- 6. Screen-solution compatibility
- 7. Sponge phase
- 8. Rational host lipid design
- 9. Lipid screening
- 10. When protein concentration is low
- 11. Cell-free expressed protein
- 12. Experimental phasing
- 13. Activity assays in meso
- 14. In meso structures
- 15. Serial crystallography
- 16. Water-soluble proteins
- 17. An evolving in meso screening strategy
- 18. Facts and figures online
- 19. Prospects
- References
- 1. Introduction
- 2. A working model for in meso crystallization
- 3. The in meso method: practical issues and challenges
- 4. High-throughput crystallogenesis and the in meso robot
- 5. Mesophase compatibility with protein-solution components
- 6. Screen-solution compatibility
- 7. Sponge phase
- 8. Rational host lipid design
- 9. Lipid screening
- 10. When protein concentration is low
- 11. Cell-free expressed protein
- 12. Experimental phasing
- 13. Activity assays in meso
- 14. In meso structures
- 15. Serial crystallography
- 16. Water-soluble proteins
- 17. An evolving in meso screening strategy
- 18. Facts and figures online
- 19. Prospects
- References
IYCr crystallization series
A comprehensive review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes
aMembrane Structural and Functional Biology Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland
*Correspondence e-mail: martin.caffrey@tcd.ie
The lipid cubic phase or in meso method is a robust approach for crystallizing membrane proteins for The uptake of the method is such that it is experiencing what can only be described as explosive growth. This timely, comprehensive and up-to-date review introduces the reader to the practice of in meso crystallogenesis, to the associated challenges and to their solutions. A model of how crystallization comes about mechanistically is presented for a more rational approach to crystallization. The possible involvement of the lamellar and inverted hexagonal phases in crystallogenesis and the application of the method to water-soluble, monotopic and lipid-anchored proteins are addressed. How to set up trials manually and automatically with a robot is introduced with reference to open-access online videos that provide a practical guide to all aspects of the method. These range from protein reconstitution to crystal harvesting from the hosting which is noted for its viscosity and stickiness. The sponge phase, as an alternative medium in which to perform crystallization, is described. The compatibility of the method with additive detergents, precipitant-screen components and materials carried along with the protein such as denaturants and reducing agents is considered. The powerful host and additive lipid-screening strategies are described along with how samples that have low protein concentration and cell-free expressed protein can be used. Assaying the protein reconstituted in the bilayer of the cubic phase for function is an important element of quality control and is detailed. Host lipid design for crystallization at low temperatures and for large proteins and complexes is outlined. Experimental phasing by heavy-atom derivatization, soaking or co-crystallization is routine and the approaches that have been implemented to date are described. An overview and a breakdown by family and function of the close to 200 published structures that have been obtained using in meso-grown crystals are given. Recommendations for conducting the screening process to give a more productive outcome are summarized. The fact that the in meso method also works with soluble proteins should not be overlooked. Recent applications of the method for in situ serial crystallography at X-ray free-electron lasers and synchrotrons are described. The review ends with a view to the future and to the bright prospects for the method, which continues to contribute to our understanding of the molecular mechanisms of some of nature's most valued proteinaceous robots.
Keywords: crystallization; lipid cubic phase; macromolecular crystallography; membrane-protein structure; mesophase; robot; structure–function; water-soluble proteins.
1. Introduction
As of this writing, there are close to 200 records in the Protein Data Bank (PDB; Berman et al., 2003; https://www.rcsb.org ) attributable to the lipid cubic phase (LCP) or in meso method of crystallizing membrane proteins (Figs. 1 and 2, Table 1). The first appeared some two decades ago in 1996. Remarkably, almost half of the records have been deposited in the PDB since the beginning of 2012. This attests to the explosive growth in the rate at which the method is being used.
|
The in meso method has had some high-profile successes of late. These include the β2-adrenergic receptor–Gs protein complex, a structure that figured prominently in the 2012 Nobel Prize in Chemistry awarded to Robert Lefkowitz and Brian Kobilka (Rasmussen, Choi et al., 2011), and channelrhodopsin, of optogenetics fame (Kato et al., 2012). Such notoriety undoubtedly contributes to interest in the method. However, a broader adoption of the method is more likely to reflect the success that it has had with an impressive range of membrane proteins and complexes. The equipment, materials and supplies needed to set up in meso crystallization are, for the most part, now available commercially, making the method more generally accessible. Also important are detailed protocols supported by open-access online instructional videos to aid the neophyte get up and running with little effort and at low cost. In the following, a comprehensive review is presented of the in meso method as applied to membrane proteins, including its recent application in the area of in situ serial crystallography. The use of the method with soluble proteins is also reviewed.
2. A working model for in meso crystallization
A proposal has been advanced for how in meso crystallogenesis takes place at the molecular level (Fig. 3; Caffrey, 2008). It typically begins with an isolated biological membrane that is treated with detergent to solubilize the target protein. The protein–detergent complex, in the form of a mixed micelle, is purified by standard wet-laboratory biochemical methods. Homogenizing with a monoacylglycerol (MAG) effects a uniform reconstitution of the purified protein into the bilayer of the cubic phase. The latter is bicontinuous in the sense that both the aqueous and bilayer compartments are continuous in three dimensions. Upon reconstitution, the protein ideally retains its native conformation and activity and has complete mobility within the plane of the cubic phase bilayer. A precipitant is added to the which triggers a local alteration in properties that include phase identity, microstructure, long-range order and Under conditions leading to crystallization, one of the separated phases is enriched in protein, which supports nucleation and progression to a bulk crystal. The hypothesis envisions a local lamellar phase that acts as a medium in which nucleation and three-dimensional crystal growth occur. Molecular-dynamics simulations highlight the hydrophobic/hydrophilic mismatch between the protein and the surrounding bilayer in the lamellar phase as a driving force for in the membrane plane (Khelashvili et al., 2012; Johner et al., 2014). The local lamellar phase also serves as a conduit or portal for proteins on their way from the cubic phase reservoir to the growing face of the crystal. Initially at least, the proteins leave the lamellar conduit and ratchet into the developing crystal to generate a layered (type I) packing of protein molecules. Given that proteins reconstitute across the bilayer of the cubic phase with no and the three-dimensional continuity of the it is possible for the resulting crystals to be polar or nonpolar. These correspond to situations in which adjacent proteins in a layer have their long-axis director oriented in the same or in opposite directions.
The proposal for how nucleation and crystal growth come about in meso relies absolutely on the three-dimensional continuity of the Under the assumption that the sample exists as a single, liquid crystallite or mono-domain, continuity ensures that the essentially acts as an infinite reservoir from which all protein molecules in the sample can end up in a bulk crystal. Neither the lamellar (Lα) nor the inverted hexagonal (HII) phases, both of which are thermodynamically accessible mesophases in lipidic systems, have three-dimensional continuity and, alone, are unlikely to support membrane-protein crystallogenesis by the in meso method.
However, it is possible to envision crystal growth that occurs by way of a local HII phase (Caffrey, 2011, 2013). Indeed, there are several crystallization conditions, such as high salt, that favour this and that support crystal growth. As is the case with the cubic and lamellar phases, the cubic and HII phases can and do co-exist (Caffrey, 1987). Transitions between the two require inter-mesophase continuity. Since bitopic and polytopic membrane proteins span the bilayer at least once, the need to remain integral to the bilayer also prevails in the HII phase. Indeed, locations where this can happen exist throughout the HII phase, specifically at points of closest contact between lipid-coated, water-filled rods. At such locations proteins can diffuse one-dimensionally along the length of the HII phase rods to associate with one another – along and between rods – first in nuclei that, in time, evolve into macroscopic crystals. As with the lamellar phase model, the cubic phase will act as a reservoir to provide a continuous supply of proteins to the growing face of the crystal. A consequence of this growth-mechanism type is that crystal packing, initially at least, will be hexagonal as opposed to layered or type I.
That the in meso method works with bitopic and polytopic proteins, having one or several membrane crossings, respectively, has been well proven. It has also been shown to support the crystallization of water-soluble proteins (§16). A mechanism for how this comes about has been presented (Caffrey, 2008). While as yet there are no examples in the literature of the method working with monotopic or lipid-anchored proteins, we can anticipate these emerging in the not too distant future. A simple mechanism for crystallization of anchored proteins could involve a form of interdigitation. Here, the acyl chains of lipid monolayers with which the protein are associated interpenetrate across the bilayer mid-plane. This would enable contact between proteins in and orthogonal to the membrane plane, facilitating three-dimensional nucleation and crystal growth. For monotopic proteins with membrane-integral domains that can contact across the bilayer mid-plane, three-dimensional nucleation and crystal growth can come about as described above for bitopic and polytopic proteins. With peripheral or `weakly' monotopic targets, and indeed for lipid-anchored proteins, a variation of the mechanism of crystallization envisioned for soluble proteins (§16) could be invoked.
Because of the proposed need for the diffusion of proteins in the bilayer and of precipitant components in the aqueous channels of the in meso. However, crystals have been seen to form within an hour, which suggests that the slowness associated with restricted diffusion can be compensated for by a reduction in dimensionality (Caffrey, 2003, 2008). The latter is a result of the protein being confined to a lipid bilayer with its long axis oriented perpendicular to the membrane plane. Thus, the number of orientations that must be sampled to effect nucleation and crystal growth is few in meso compared with its in surfo counterpart that employs micelles and in which all of three-dimensional space is accessible.
the expectation is that crystal-growth rates might be tardyThat crystal growth takes place in a ). Settling of crystals and subsequent growth into one another are also avoided under these conditions, as is the likelihood that impurities are wafted in from the surrounding solution to poison the face of the crystal and limit growth. For all these reasons in meso crystallogenesis is similar to crystallization in space, with the prospect of producing high-quality, structure-grade crystals.
implies that it is happening in a convection-free environment. This is analogous to growth under conditions of microgravity or in a gel or a micro-fluidic channel, which offer the advantage of a stable zone of depletion around the growing crystal and thus slower and more orderly growth (Caffrey, 20003. The in meso method: practical issues and challenges
Setting up an in meso crystallization trial is straightforward (Fig. 4). Typically, it involves combining two parts protein solution with three parts lipid at 20°C (Caffrey & Cherezov, 2009; Caffrey & Porter, 2010). The most commonly used lipid is the monoacylglycerol (MAG) monoolein. According to the monoolein–water temperature–composition phase diagram (Fig. 5; Qiu & Caffrey, 2000), and assuming there is no major influence on the phase behaviour of the protein-solution components, this mixing process should generate, by spontaneous self-assembly, the cubic at or close to full hydration. The original method for mixing lipid and protein solution involved multiple, cumbersome centrifugations in small glass tubes. Harvesting crystals required cutting the tubes and searching for small crystals through curved glass, which was not easy, very inefficient and required experience, time and patience.
The cubic phase is sticky and viscous in the manner of thick toothpaste (Fig. 6). As such, it is not easy to handle. In the course of earlier lipid-phase science work carried out in the Membrane Structural and Functional Biology (MS&FB) group, we had developed tools and procedures for manipulating such refractory materials. One of these, the coupled-syringe mixing device (Fig. 4; Cheng et al., 1998), was ideally suited to the task of combining microlitre volumes of monoolein with membrane-protein solution in a way that produces protein-laden for direct use in crystallization trials with minimal waste. The mixer consists of two, positive-displacement Hamilton micro-syringes connected by a narrow-bore coupler. Lipid is placed in one syringe and protein solution in the other. Mixing is achieved by repeatedly moving the contents of the two syringes back and forth through the coupler (Caffrey & Porter, 2010). The coupler is replaced by a needle for convenient dispensing of the homogenous into wells of custom-designed, glass sandwich crystallization plates (Cherezov & Caffrey, 2003; Cherezov et al., 2004). Precipitant solutions of varying compositions are placed over the and the wells are sealed with a cover glass. For initial screening, the plates are incubated at 20°C and monitored for crystal growth. The optical quality is the best it can be given that the is held between two glass plates and the itself is transparent (Fig. 7). This means that crystals of just a few micrometres in size can readily be seen by microscope whether the proteins are coloured or not. The use of cross-polarizers can enhance the visibility of small crystals, which usually appear birefringent on a dark background; the cubic phase itself is optically isotropic and non-birefringent. An added feature of the glass sandwich plates is that the double-sided tape used to create the wells provides almost hermetic sealing, ensuring minimal change in well composition during the course of trials that can last for months. Step-by-step instructions, complete with an open-access online video demonstration of the entire in meso crystallization process, have been published (Caffrey & Cherezov, 2009; Caffrey & Porter, 2010; Li, Boland, Aragão et al., 2012; Li, Boland, Walsh et al., 2012).
4. High-throughput crystallogenesis and the in meso robot
The protocol just described refers to the manual mode of setting up crystallization trials. Accurate and precise delivery of the protein-laden e; Cherezov & Caffrey, 2005, 2006; Caffrey, Eifert, 2014). The smaller volumes mean that the in meso method works with miniscule quantities of target protein. Thus, extensive crystallization trials can be set up with just a few micrograms of valuable membrane protein, making the in meso method one of the most efficient in terms of protein (and lipid and ligands, as appropriate) requirement.
in volumes that range from picolitres to microlitres was made possible by the use of an inexpensive repeat dispenser in combination with differently sized micro-syringes (Fig. 4Whilst the repeat dispenser greatly facilitated the in meso method, it was still a manual setup with limits to the number of trials that one person could comfortably and reproducibly set up at a sitting. The need to automate the process was obvious. With the assistance of A. Peddi and Y. Zheng, engineers at The Ohio State University where the original work was carried out, we were able to perform a proof-of-principle robotics exercise employing LabView-controlled motorized translation stages operating and supporting a micro-syringe and a crystallization plate. The prototype was used to demonstrate that the viscous could be dispensed automatically and wells filled in such a way that eventually yielded crystals. This was sufficient to secure funding for a robot, which was custom-designed and built to our specifications (Cherezov et al., 2004).
The in meso robot has two arms programmed to move simultaneously. One dispenses the viscous protein-laden while the other dispenses precipitant. Typical volumes used are 30–50 nl (consisting of 12–20 nl protein solution and 18–30 nl monoolein) and 600–800 nl precipitant solution. Custom 96-well glass sandwich plates were designed which take several minutes to fill using an eight-tip robot. The robot enables the precise and accurate setting up of in meso crystallization trials with picolitre to microlitre volumes of in high-throughput mode and, if required, under challenging conditions of reduced temperature and controlled lighting. Given the success of the in meso robot, several are currently in use in laboratories throughout the world. Variations on the original design, in which tip alignment is performed automatically and in which precipitant is handled by disposable tips, are now commercially available. Another uses a 96-tip liquid-dispensing head to deliver precipitant solution in a single action, thereby reducing the time taken to set up a single plate to less than 2 min. These and other commercially available robots represent important advances that simplify the in meso setup and make the method more generally available and user-friendly.
With the success that the in meso method has had, it is perhaps not unexpected to find products appearing on the market in support of this now proven, robust crystallogenesis approach. In addition to the in meso robots, these include a number of precipitant screen kits, glass and plastic sandwich plates, and a plate that comes complete with lipid-coated wells. The vendors indicate that the latter can be used with a liquid-dispensing robot for protein-solution delivery first and precipitant post-swelling.
5. compatibility with protein-solution components
As alluded to above, what happens during in meso crystallization is intimately tied up with behaviour (Fig. 3; Caffrey, 2008). The working hypothesis for how nucleation comes about begins with the protein reconstituting into the continuous bilayer of the cubic phase. Precipitant is added, which triggers the local formation of a lamellar phase into which the protein preferentially partitions and concentrates in a process that leads to nucleation and crystal growth. Experimental evidence in support of aspects of this model has been reported (Cherezov & Caffrey, 2007; Caffrey, 2008).
Experience built up over several years of working with the in meso method suggests that the behaviour observed during the course of crystallization mimics that of the monoolein–water system (Fig. 5). The implication therefore is that the protein solution has little effect on the behaviour of the hosting lipid into which the protein is reconstituted. This solution, along with the target protein, typically includes lipid, detergent, buffers and salt at a minimum. Other components, such as glycerol, sulfhydryl reagents, denaturants etc., are not uncommon. Each of these can impact on phase behaviour and, by extension, the outcome of a crystallization trial. In the interests of learning about component compatibility, the sensitivity of the monoolein–water cubic phase system to their inclusion has been evaluated. Our findings indicate that the default cubic is remarkably resilient and retains its phase identity and microstructure in the presence of a vast array of different additives. These include glycerolipids, cholesterol, free detergents, denaturants, glycerol and sulfhydryl reagents, among others (Ai & Caffrey, 2000; Cherezov et al., 2001, 2002; Misquitta & Caffrey, 2003; Clogston & Caffrey, 2005; Clogston et al., 2005; Liu & Caffrey, 2005, 2006; Cherezov, Clogston et al., 2006; Cherezov, Yamashita et al., 2006). Of course, for each there is a concentration beyond which the cubic phase is no longer stable. In most cases, these limits have been identified.
Occasionally, the concentration of a protein-solution component is not known exactly. Detergent is a case in point. This poses a problem because if there is too much detergent the bulk lamellar phase may form but will not support crystallization (Ai & Caffrey, 2000; Misquitta & Caffrey, 2003). It may also be that a new detergent is being used whose compatibility with the cubic phase is not known. In this case, a small amount of the buffer employed to solubilize the protein or, preferably, the protein solution itself can be used to prepare the The physical texture, appearance between crossed polarizers and/or small-angle X-ray scattering (SAXS) behaviour of the will indicate which phase has been accessed. If, for example, it is a lamellar phase that forms, suggesting too much detergent, then another purification step in which its concentration in the final protein solution is reduced may be sufficient to solve the problem. We have encountered situations with bacteriorhodopsin where the particular preparation ended up having an excess of detergent. The first formed was lamellar, but when it was used in combination with certain precipitants a transition back to the cubic phase was induced and the sample went on to grow crystals (Misquitta & Caffrey, 2003; Caffrey, 2008). This highlights the importance of understanding behaviour for more rational and productive crystallization.
6. Screen-solution compatibility
As noted, in meso crystallization relies on a bicontinuous which acts as a reservoir to feed protein into nucleation sites and for crystal growth. Crystallization screening requires that chemical space be interrogated over wide limits to find conditions that support crystallogenesis. In the screening process, therefore, the protein-laden is exposed to precipitant solutions that encompass hundreds and perhaps thousands of different chemical compositions. Screen-solution components typically include buffers that cover a wide pH range, polymers, salts, small organics, detergents, apolar solvents, amphiphiles etc., and all at different concentrations. Each component can potentially destabilize the In a separate study using SAXS, we examined the compatibility of the default monoolein–water cubic phase with various commonly used precipitant screen solutions (Cherezov et al., 2001). What we found was hardly surprising. Compatibility was temperature-dependent and the usual suspects, which included organic solvents, destroyed the cubic phase, rendering these screen solutions effectively useless. A goal of the study was to design screens that were mesophase-friendly. However, this goal was never pursued; instead, we have opted for the convenience of commercial screen kits mindful of the fact that certain conditions are not useful. As a result, certain kits are simply not used because they contain too few conditions that are compatible with the cubic phase.
7. Sponge phase
During the course of ), thereby enabling the to absorb more lyotrope (aqueous solution). This is evident in the SAXS pattern, where the lattice parameter of the cubic phase rises with spongifier concentration. Eventually, the loses order and the low-angle diffraction pattern becomes diffuse. Fortunately, the sponge phase retains its bicontinuity and, as a result, can support in meso crystallogenesis (Cherezov, Clogston et al., 2006; Caffrey, 2008; Wöhri et al., 2008). One advantage of the sponge phase is that its aqueous channels are dilated. Thus, proteins with large extramembrane domains should be accommodated in and amenable to crystallogenesis from the sponge phase (§8.2). Further, the reduced interfacial curvature and bending rigidity are likely to facilitate more rapid and long-range diffusion within the lipid bilayer. Since net movement of protein from the reservoir to sites is a requirement for crystallization, this effect alone should contribute to improved crystallization, provided, of course, that the process is not too fast. Interestingly, many of the proteins that have yielded to the in meso method have been crystallized under conditions that favour sponge-phase formation (Table 1; Caffrey et al., 2012).
compatibility studies, we noticed that particular screen components caused the cubic phase to `swell' and, under certain conditions, to form what is referred to as the sponge phase. The latter evolves from the cubic phase as a result of the `spongifying' component lowering the bilayer interfacial curvature and presumably its bending rigidity (Chung & Caffrey, 1994Reflecting the utility of the sponge phase for in meso crystallogenesis, a number of commercial screening kits now include spongifiers such as polyethylene glycol (PEG), Jeffamine and butanediol, among others. Some of these provide a preformed sponge phase to which the protein solution is added directly. We continue to use the original method that involves an active protein-reconstitution step and where the entire crystallization screen space is available for sampling.
8. Rational host lipid design
8.1. Low-temperature crystallogenesis
The MS&FB group has devoted considerable time and effort to establishing rules for rationally designing in meso crystallogenesis at low temperatures. Certain proteins are labile and require handling at low temperatures. A potential problem with the in meso method, in the default mode at least, is that it relies upon monoolein as the hosting lipid. The cubic phase formed by monoolein is not thermodynamically stable below about 17°C (Qiu & Caffrey, 2000) and performing crystallization trials in a cold room at 4–6°C is risky. For this low-temperature application, therefore, a cis-monounsaturated monoacylglycerol, 7.9 MAG, was designed using the rules referred to above. The target MAG was synthesized and purified in-house and its phase behaviour was mapped out using SAXS (Misquitta, Cherezov et al., 2004). As designed, it produced a cubic phase stable in the range from 6 to 85°C, as designed. 7.9 MAG has been used in the crystallization of a number of membrane proteins in the MS&FB group and beyond. It, along with other synthetic and natural MAGs (see below), are available to the community by way of commercial vendors.
with specific end uses. One such application concerned the development of a host lipid for use inThe word `risky' was used in the previous paragraph in reference to low-temperature crystallization with monoolein as the hosting lipid. This reflects the fact that it is possible to perform successful in meso work with monoolein at 4°C provided that the system undercools. Fortunately, the cubic phase is noted for this capacity (Fig. 5; Briggs & Caffrey, 1994; Qiu & Caffrey, 2000), and we regularly perform successful crystallization trials with monoolein in the 4–17°C range. As expected, under these metastable conditions the will occasionally convert to the lamellar crystalline or solid phase, which is useless as far as crystallization is concerned.
Sugar-phytane et al., 2004) and that might find application for in meso crystallization at reduced temperatures.
have been synthesized that form the fully hydrated cubic phase in the 10–70°C range (Hato8.2. Proteins and complexes with large membrane footprints and large extramembrane domains
In the following, two recent examples of caa3 oxidase from Thermus thermophilus (Lyons et al., 2012). This terminal oxidase is a large 120 kDa heterotrimeric protein with 23 transmembrane helices and a cytochrome c-like extramembrane domain as a C-terminal extension to one of its subunits. Extensive crystallization trials using traditional vapour-diffusion methods failed to produce structure-grade crystals. The in meso method was considered as an appropriate alternative. At the time that the study was undertaken, in meso crystallization had generated crystals and a structure of light-harvesting complex II, LHII, whose bulk in the plane of the membrane resembled that expected for caa3. Initial in meso trials with the default lipid, 9.9 MAG or monoolein, failed to produce useful crystals. Anticipating the likelihood that 9.9 MAG would not suit every membrane protein, the lipid-synthesis program in the MS&FB Group (Caffrey et al., 2009; Yang et al., 2012) provided alternative MAGs with which to screen for crystallogenesis. The first of these tested was 7.7 MAG, which has an acyl chain 14 carbon atoms long and a cis double bond between carbon atoms 7 and 8. 7.7 MAG had been shown to form a cubic with a thinner, less highly curved bilayer and with enlarged aqueous channels (Misquitta, Misquitta et al., 2004). A thinner bilayer was considered to be desirable for use with caa3 because it more suitably complemented the hydrophobic thickness predicted for related cytochrome oxidases of known structure. Additionally, the larger aqueous channels provided by 7.7 MAG were attractive in the context of caa3 with its added extramembrane bulk in the form of a cupredoxin and the aforementioned tethered cytochrome-c-like domain. As expected, 7.7 MAG produced crystals; upon optimization they provided a structure at 2.36 Å resolution (Lyons et al., 2012).
rationally designed for use in crystallizing targets with large footprints in the plane of the membrane and/or extensive extramembrane domains are described. The first refers to cytochromeThe second example is the β2-adrenergic receptor–Gs protein complex (Rasmussen, DeVree et al., 2011). Earlier work had shown that the receptor alone produced structures to high resolution with the default lipid 9.9 MAG using the in meso method. However, efforts to grow structure-grade crystals of the receptor as a complex with its cognate Gs protein in monoolein failed. The Gs protein is itself a large heterotrimeric complex with a molecular weight of ∼80 kDa. It binds to the exposed intracellular surface of the receptor and adds considerable extramembrane bulk to the target. In this particular instance the Gs protein had bound to it a camelid single-chain antibody or nanobody (15 kDa), and T4 lysozyme (19 kDa) was fused to the N-terminus of the receptor. Both contributed additional extramembrane heft to the complex. Given that the cubic phase prepared with monoolein alone has aqueous channels in which the water-soluble domains must reside that are only 50 Å in diameter, failure to crystallize in monoolein did not come as a surprise. 7.7 MAG, with its significantly larger aqueous channels, was immediately identified as a suitable alternative host lipid and, with some limited optimization, it generated diffraction-quality crystals and a structure of the complex (Rasmussen, DeVree et al., 2011). Interestingly, the precipitant used for the production of the final crystals included PEG 400, a known spongifier, and the crystals were harvested from what appeared to be a sponge phase. It seems likely therefore that the short-chain MAG and the spongifier worked hand in hand to generate a bicontinuous medium that accommodated unrestricted diffusion and that facilitated crystallization of the complex with its extensive extramembrane domains. Future in meso crystallization trials with targets of this sort will undoubtedly benefit from the use of alternative MAGs in concert with sponge phase-inducing precipitants. Commercial crystallization kits that include such materials are likely to be forthcoming. It is important to note that in all of the aforementioned GPCR work the host MAG was doped with cholesterol (see the following section).
9. Lipid screening
9.1. Additive lipid
Early on in the development of the in meso method, the author recognized that monoolein, as the lipid used to create the hosting is a most uncommon membrane lipid. The sense was that this lipid might rightly be regarded as foreign by certain target proteins and cause them to destabilize or to adopt an unnatural conformation. One possible solution was to use a native membrane lipid that would form the requisite cubic phase at 20°C. None were available. An alternative was to use monoolein, or another suitable MAG, as the hosting lipid and to augment it with typical membrane thereby creating a more native-like environment. Accordingly, the carrying capacity of the monoolein cubic phase for a number of different was established using SAXS (Cherezov et al., 2002). This capacity amounted to about 20 mol% in the cases of phosphatidylcholine, phosphadidylethanolamine and cholesterol, with lesser amounts of phosphatidylserine and cardiolipin being accommodated. The approach of using additive has had spectacular successes in the GPCR field, where cholesterol doping of the cubic phase was and continues to be critical to the production of structure-grade crystals (Caffrey et al., 2012). Recently, it proved to be crucial in obtaining a high-resolution structure of human microsomal prostaglandin E2 synthase 1 (mPGES1), where the host lipid, 8.8 MAG, was doped with 2 mol% DOPC (Li et al., 2014).
9.2. Host lipid
Monoolein was the first lipid used for in meso crystallogenesis. From the outset, it was recognized that this one lipid may not work with all target membrane proteins (Caffrey, 2003; Misquitta, Cherezov et al., 2004). These, in turn, come from a variety of native membranes which differ in lipid composition, surface charge and packing density, fluidity and polarity profile, bilayer thickness, intrinsic curvature, bending elasticity, etc. Thus, having a range of MAGs that differ in acyl-chain characteristics with which to screen was deemed to be important. Using principles of rational design, several MAGs were identified with the requirement that they form the cubic phase at 20°C under conditions of full hydration. A number of meeting this specification have been synthesized and characterized in-house. They constitute a very successful host-lipid screen in the MS&FB group and beyond. With several targets, which include β-barrels, α-helical proteins, a GPCR–Gs protein complex and an integral peptide antibiotic, crystals have been grown by the in meso method using these alternative MAGs (Misquitta, Cherezov et al., 2004; Misquitta, Misquitta et al., 2004; Höfer, Aragão, Lyons et al., 2011; Li et al., 2011, 2015; Li, Shah et al., 2013; Ring et al., 2013; Lyons et al., 2014; Malinauskaite et al., 2014; Takeda et al., 2014). In a number of cases monoolein either failed to produce crystals or the crystals that it did produce were not of diffraction quality. It was only when MAGs from the host-lipid screen were used that structure-grade crystals were obtained. A number of these novel MAGs are available to the community through commercial suppliers.
10. When protein concentration is low
The driving force for nucleation is greater the more supersaturated the system is. Thus, a common strategy in the area of crystallization is to work at the highest possible protein concentration to favour nucleation, and to lower the protein concentration subsequently to just above the (super)solubility limit for the slow, orderly growth of a few, good-quality crystals. It is likely that the same principles apply to crystallization in meso, where initially the highest possible protein concentration should be used in support of nucleation. There are at least two issues that must be dealt with in this context that apply to membrane proteins. Firstly, most membrane proteins are prepared and purified in combination with detergents. Thus, the detergent is carried along with the protein into the crystallization mixture. It follows then that as the protein concentration increases, the detergent concentration will rise in parallel. This may work against crystallization because high levels of detergent can destabilize the host (§5; Ai & Caffrey, 2000; Misquitta & Caffrey, 2003; Caffrey, 2008). Of course, the sensitivity to added detergent will depend, among other things, on the identities of the host lipid and detergent. Completely removing the detergent before folding the protein into the crystallization mixture is usually not an option because it is commonly required to keep the protein soluble as a mixed micelle. One alternative is to reduce the detergent load to an acceptable level before combining the protein with the host lipid. This can be performed with BioBeads or by eluting the protein in a highly concentrated form from an affinity column. Using detergents with low values, such as lauryl maltose neopentyl glycol (MNG-DDM), is also worth investigating.
Raising the protein concentration in the solution used to make the 5; Li & Caffrey, 2014). If this approach is used, however, the additive should be removed or its concentration dramatically reduced prior to running in meso crystallization trials. Simply equilibrating the thus formed with excess precipitant under standard crystallization conditions (50 nl + 800 nl precipitant) will eventually reduce the additive concentration by 40-fold. Further reductions are possible following the protocol described in the next paragraph.
without simultaneously elevating the detergent concentration is an important goal to work towards. This can be approached by selecting only the peak fractions from a final polishing gel-filtration column and using the largest workable molecular-weight cutoff filters for protein concentration. Glycerol and urea can raise protein solubility and both are compatible with the cubic phase (§The second issue has to do with raising the concentration of protein in the lipid bilayer of the cubic phase to facilitate nucleation. Two approaches can be tried that are quite different but that achieve the same end. The first exploits the water-carrying capacity of the cubic phase, a property that varies with lipid identity (Misquitta, Misquitta et al., 2004; Caffrey, 2008). Thus, the reconstituted protein will be more concentrated in the bilayer of a cubic phase prepared with a lipid of high water-carrying capacity than would be obtained for a less hydrating lipid. The second approach, referred to as the `cubicon' method, involves sequential reconstitutions in which the protein concentration in the bilayer rises in each round (Li & Caffrey, 2014). The membrane protein preferentially partitions from the aqueous solution into the bilayer of the cubic phase. If the reconstitution step is repeated using a single bolus and with a series of solutions of protein at low concentration, the protein load in the bilayer of the will increase in each reconstitution round, leaving excess aqueous solution depleted of protein. This protein-depleted solution is removed before the next round of reconstitution commences. Successful applications of the cubicon approach have been implemented in the author's laboratory for several integral membrane-protein targets (Li & Caffrey, 2014; P. Ma & M. Caffrey, unpublished work).
11. Cell-free expressed protein
For the most part, in meso crystallization trials are conducted with naturally abundant proteins or proteins produced recombinantly in expression systems such as Escherichia coli, insect or mammalian cells. Cell-free expression is a method with considerable promise in the membrane-protein field (Schwarz et al., 2007). It is easy to perform, milligram quantities of protein can be produced overnight and costs are reasonable. Because the system is open, labelling (with selenomethionine, for example) is simple, harvesting protein is straightforward and the newly synthesized protein is already relatively pure. The cell-free method has been used to express integral membrane proteins for To date, however, only three have been crystallized in surfo, two of which have yielded crystal structures (Chen et al., 2007; Wada et al., 2011). These include the transporter EmrE at 3.8 Å resolution and a light-driven pump at 3.2 Å resolution. Therefore, while the method is proven, and indeed kits for in vitro expression are available commercially, it cannot be considered to be routine. Intrigued by what the cell-free method had to offer with regard to quality protein for structure work, we evaluated its applicability using the in meso crystallogenesis method with diacylglycerol kinase (DgkA) as a test protein. It worked spectacularly well. Milligram quantities of the kinase were produced overnight as aggregated protein, the protein was solubilized, reconstituted into the cubic phase and crystallized. Satisfyingly, the structure, solved to 2.3 Å resolution with little optimization of crystallization conditions, was remarkably similar to that of in vivo-produced protein (Boland et al., 2014).
In the DgkA study just described, we chose to carry out expression in the absence of a membrane mimetic in part because the aggregated protein thus formed in vitro resembled the inclusion bodies that the protein overexpressed in vivo forms naturally and that had been used successfully for crystallography. However, it is possible to perform cell-free expression in the presence of a membrane mimetic for the direct production of functional protein. To date, detergent micelles, liposomes, nanodiscs and bicelles have been used for this purpose, and each has its own pros and cons. A logical extension to this approach is to use the bicontinuous lipid as an alternative receiving membrane mimetic with several attractive features. To begin with, the cubic phase comprises an essentially limitless reservoir for the expressed membrane protein throughout which it can diffuse. Secondly, the includes a familiar bilayered membrane in which the newly synthesized protein is likely to feel at home and to retain a native, functional fold. Thirdly, the bicontinuous nature of the means that both sides of the membrane-embedded protein are accessible, which is important for functional characterization and assay development. Fourthly, should certain proteins prove refractory to unaided insertion into the translocon proteins can be added to facilitate the process. Fifthly, because of its sticky and viscous nature the is readily harvested for subsequent use as a system with which to perform biochemical, pharmacological and biophysical characterization. Finally, another consequence of the unique rheological properties of the is that it lends itself to miniaturization and to microarray-type applications for high-throughput screening. One of our immediate objectives is to use the protein-laden for direct in meso crystallization. Thus, by performing cell-free expression and in meso crystallization in tandem, the need to separately isolate and purify the protein is avoided, rendering the process from gene to crystal highly efficient in terms of time and cost whilst eliminating the potential damaging effects of solubilizing detergents.
12. Experimental phasing
The structures solved using in meso-grown crystals have relied predominantly on for phasing. The challenges associated with experimental phasing derive, in part, from a low anomalous signal-to-noise ratio owing to a combination of background low-angle and wide-angle scatter from adhering and the need to work with small and sometimes poorly diffracting, radiation-sensitive crystals. As often as not, data must be collected in angular wedges from different parts of a single crystal or from multiple crystals, and merging data satisfactorily can be a challenge. Despite the difficulties, successes with experimental phasing have been reported. In the past three years alone, the following structures have been solved by this method: channelrhodopsin from Chlamydomonas reinhardtii (PDB entry 3ug9 ; mercury-MAD; Kato et al., 2012); the Na+/Ca2+ exchanger from Methanococcus jannaschii (PDB entry 3v5u ; samarium-SAD; Liao et al., 2012); β-barrels from E. coli (PDB entry 4e1s ; selenomethionine-SAD; Fairman et al., 2012) and Y. pseudotuberculosis (PDB entry 4e1t ; selenomethionine-SAD; Fairman et al., 2012); DgkA from E. coli (PDB entry 3ze3 ; selenomethionine-SAD; Li, Lyons et al., 2013); human mPGES1 (PDB entry 4bpm ; sulfur-SAD; Li et al., 2014); CDP-alcohol phosphotransferase (PDB entry 4o6m ; selenomethionine-SAD; Sciara et al., 2014); and claudin-15 (PDB entry 4p79 ; selenomethionine-MAD; Suzuki et al., 2014). It would appear therefore that whilst challenging, experimental phasing is a reality and should not limit using crystals grown in meso. Indeed, with careful measurements, native sulfur-SAD phasing with a single in meso crystal is possible (Weinert et al., 2014). A detailed case study of experimental phasing as applied to DgkA has recently been reported (Li et al., 2015).
13. Activity assays in meso
It is assumed that proteins reconstituted prior to crystallization are functional in meso. In the case of BtuB, this was examined by measuring substrate (cyanocobalamin; CNCbl) binding to the protein incorporated into the cubic phase (Cherezov, Yamashita et al., 2006). Protein-free control samples exhibited no binding, whereas test in meso BtuB-containing samples displayed convincing visual evidence of substrate uptake (CNCbl is pink). Binding was shown by quenching of the intrinsic fluorescence of aromatic residues by CNCbl and by direct ligand binding to be tight, with an apparent Kd value of ∼1 nM. Similar Kd values have been reported for the native membrane-bound and micellarized form of the protein. Sialic acid binding to the adhesin OpcA, measured by fluorescence quenching as with BtuB, was identical in meso and in detergent solution (Cherezov et al., 2008). Taken together, the data support the view that these β-barrel proteins reconstitute into the bilayer of the cubic phase in an active form prior to in meso crystallization.
Functional activity assays in meso have been extended to include membrane-protein enzymes (Li & Caffrey, 2011). In the case of DgkA, a coupled-enzyme assay was used. With phosphatidylglycerol phosphate synthase (PgsA), activity was quantified by direct assay. In both cases, the viscous, sticky and porous nature of the cubic phase was used to advantage in enabling spectrophotometric activity assays to be performed in a high-throughput multi-well microplate format. With both enzymes, the cubic served as a useful and a convenient nanoporous membrane mimetic that supported native-like activity.
Recent studies with the dopamine 2 long (D2L) and histamine 1 (H1) GPCRs indicate ligand binding in the nanomolar range based on radioligand assays (Darmanin et al., 2012). In this study, the receptors were reconstituted into the cubic phase by a passive method and showed significantly enhanced specific binding compared with their detergent-solubilized counterparts.
14. In meso structures
As of this writing, the in meso method accounts for almost 200 records in the PDB that relate to integral membrane proteins and (https://www.pdb.org ; Fig. 1, Table 1). This corresponds to about 10% of all published membrane-protein structures, representing a wide range of distinct membrane-protein types, sizes and oligomeric forms. With successes that include bacterial rhodopsins, light-harvesting complex II, photosynthetic reaction centres, β-barrels, GPCRs and a GPCR–G protein complex, transporters, channels, enzymes, cytochrome oxidases, channelrhodopsin, a membrane-protein insertase, tight-junction claudin-15 and an integral membrane peptide, the method has a convincing record of versatility and range. Each of these membrane-protein types represents larger families, the members of which become suitable candidates for in meso crystallogenesis. The GPCR family is a case in point, with almost 800 distinct GPCRs coded for in the human genome alone. Accordingly, the in meso method, in combination with the necessary protein-engineering and receptor-stabilization strategies, is now contributing to the generation of GPCR structures in what amounts to a production-line fashion. Evidence in support of this statement is the recent spate of receptor structures, almost 40 in the past two and a half years, courtesy of the in meso method. The same degree of success can be anticipated for other membrane-protein families. Transporters would appear to be moving in this direction (Fig. 1, Table 1).
The further development of the in meso crystallogenesis approach is an important goal for members of the MS&FB group. One direction this has taken concerns the utility of the method with small membrane proteins. A separate analysis performed using a model cubic phase under restricted conditions indicated that suitable targets would need to include at least five transmembrane helices (Grabe et al., 2003). Our experience with the sponge-phase variant of the cubic phase suggested otherwise. Accordingly, the utility of the method with a `mini-protein', the pentadecapeptide antibiotic linear gramicidin, was investigated. It worked remarkably well, providing a structure with a resolution of better than 1.1 Å (Höfer et al., 2010; Höfer, Aragão & Caffrey, 2011; Höfer, Aragão, Lyons et al., 2011). This result is significant because it highlights the utility of the method with proteins having small footprints in the plane of the membrane. which abound in nature and include a multitude of receptors and signalling proteins.
15. Serial crystallography
15.1. With free-electron laser X-rays
Serial femtosecond X-ray crystallography (SFX) is a relatively new method for collecting crystallographic information from small crystals fed into a free-electron laser (FEL) beam composed of high-fluence X-ray bunches mere femtoseconds long (Chapman et al., 2011, 2014; Spence et al., 2012). Each encounter between an X-ray bunch and a microcrystal (hit) ideally gives rise to a single, still diffraction pattern with greater than ten measurable reflections. Since the crystals are randomly oriented, collecting patterns from enough of them (typically many thousands) produces a complete data set of high redundancy for to date by with just one exception (Barends et al., 2014). Data are typically collected in an evacuated interaction sample chamber operated at 20°C. Despite the intensity of the X-ray bunch (∼1012 photons per bunch), each is of such short duration that insufficient time (the pristinity window) is available for the changes associated with radiation damage to progress sufficiently before the diffracted X-rays have departed (run) with their structural manifest to be recorded. This is referred to as `hit and run' (Caffrey, Li et al., 2014) or `diffraction before destruction' SFX (Chapman et al., 2014).
Until recently, a fluid medium had been used to ferry crystals of membrane proteins across the beam for SFX (Chapman et al., 2011; Johansson et al., 2012). The process involved voluminous flow rates. Because productive interactions between X-rays and crystals in the flowing stream were so infrequent, vast amounts of valuable membrane protein were required for data collection and most of the protein went to waste. Typically, only one in 25 000 crystals produced a useful diffraction pattern. Thus, for example, when photosystem I (PSI) crystals were used dispersed in a liquid jet, data collection required 10 mg of protein (Chapman et al., 2011). By contrast, when photosynthetic reaction centre crystals were delivered dispersed in the more viscous lipid sponge phase, 3 mg of protein were needed (Johansson et al., 2012). The idea was subsequently mooted that using the highly viscous LCP, in which the membrane-protein crystals can be grown by the in meso method, might provide a transport medium for SFX with better hit rates. As a result of being so viscous, the flow rate would be reduced dramatically. If high enough crystal densities in the LCP could be achieved, the rate of delivery of crystals and X-rays to the interaction region could be matched for a most efficacious use of both. The method is hereafter referred to as LCP-SFX.
LCP-SFX is appealing as a method because it offers the prospect of obviating some of the issues that arise with in meso-based using synchrotron X-radiation. With the in meso method, crystals are typically grown in a sealed glass sandwich plate. Harvesting crystals is a somewhat cumbersome process that can lead to substantial loss of crystals and to degradation in diffraction quality. Data collection at a synchrotron source is typically performed at 100 K. Such a frigid temperature can stabilize conformational substates, particularly in the side chains of the protein, that are not physiologically relevant and that are possibly misleading as far as functional interpretation is concerned (Fraser et al., 2011). Radiation damage is also a major concern with synchrotron-radiation sources, where residues such as aspartate and glutamate are particularly prone to undergo decarboxylation (Burmeister, 2000). Damage can be mitigated to a degree with large crystals, beam attenuation and data collection at cryo-temperatures, often requiring many tens of crystals. In this context, LCP-SFX was attractive in that it offered what amounts to in situ data collection with micrometre- or nanometre-sized crystals at or close to the more physiologically relevant 20°C and the prospect of outrunning radiation damage.
A test of the proposed LCP-SFX idea was performed at the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS) over the course of seven 12 h data-collection shifts in March 2013. Diffraction data were collected on Cornell–SLAC Pixel Array Detector (CSPAD) detectors. Crystals were ported across the XFEL beam as a continuously extruded bolus of et al., 2014). The feasibility study was spectacularly successful, yielding high-resolution structures for three integral membrane proteins that included diacylglycerol kinase (DgkA) and two GPCRs (Liu et al., 2013, 2014; Caffrey, Li et al., 2014); a fourth is in the works. Record low quantities of protein were needed to obtain structures. In the case of DgkA this amounted to just 220 µg protein and 42 µl cubic phase. Clearly, the method is on deck for use in novel ways with a host of other membrane proteins and complexes.
by means of a specially engineered LCP injector (Weierstall15.2. With synchrotron X-rays
The LCP injector developed for SFX (§15.1; Weierstall et al., 2014) mimics the gas-tight Hamilton micro-syringes used in the coupled-syringe mixing device and for dispensing into the wells of crystallization plates (Cheng et al., 1998; Cherezov & Caffrey, 2005; §3). However, the LCP injector was designed to operate at much higher pressures and therefore can, in a leak-free manner, extrude the viscous, microcrystal-laden through a long, narrow-bore capillary for SFX. The problem with SFX measurements, however, is that XFEL facilities are in very short supply globally and are in great demand. It made sense therefore to look to other, more readily available bright X-ray sources, such as synchrotrons, with which to make use of the LCP injector. To date, successful tests of the injector have been carried out with crystals of lysozyme and of integral membrane proteins, and data of sufficient quantity and quality have been collected for (https://www.esrf.eu/home/news/general/content-news/general/novel-injector-allows-X-rays-to-map-membrane-proteins.html ). Like SFX, this amounts to serial crystallography (SX) where data must be collected from thousands of micro-crystals jetted across the beam. The advantages of this approach include the fact that synchrotron beam time is more generally available and accessible, and that measurements are made on the naked without attenuating and scattering windows, in air under close to in situ conditions, obviating the need for cumbersome, inefficient harvesting, and at the more physiologically relevant room temperature. However, there are challenges that include radiation damage, injector maintenance and operation by skilled personnel, and the need for a high density of micrometre-sized crystals in a that is dust-free to prevent injector clogging. It is early days in the development of this type of SX methodology. Time will tell whether it offers real advantages over other methods of data collection. If it does, it will take the pressure off XFEL sources, which can be used in applications for which they are uniquely suited.
16. Water-soluble proteins
The in meso method was developed and is used primarily for crystallizing membrane proteins. However, it also works with soluble proteins. Lysozyme, insulin, α-lactalbumin and thaumatin are cases in point (Landau et al., 1997; Cherezov et al., 2004; Tanaka et al., 2004; Aherne et al., 2012). There may well be advantages to growing soluble protein crystals in meso that relate to the fact that it mimics crystallization in gels and under conditions of microgravity (§2; Caffrey, 2003). Such conditions stabilize the depletion zone and minimize the settling of crystals on top of one another and the wafting of contaminants to the growing surface of the crystal, all of which are associated with improved crystal quality.
A quick and easy protocol for crystallizing lysozyme by the in meso method, which gives 15–20 µm-sized crystals within an hour, has been developed (Aherne et al., 2012). It is currently being used for instructional purposes and as a training tool. Increasingly, the approach is being used to test new applications of the cubic phase: for example, as a viscous, slow-`flowing' medium in which to port microcrystals of soluble proteins and complexes into an XFEL or synchrotron beam for efficient, high hit-rate SFX or SX (Caffrey, Li et al., 2014; Liu et al., 2014). Crystals can be grown in situ and used essentially as with membrane proteins. An alternative approach is to combine extant crystals with pre-formed to create a dispersion that can be loaded directly into the reservoir of the LCP injector for SFX or SX measurements. In the latter case, the would best be prepared with the mother liquor in which the soluble protein crystals grew. As with membrane proteins, MAGs with different acyl-chain characteristics and correspondingly different microstructures, transport properties and rheologies should prove to be useful for generating and porting crystals of the widest possible range of soluble protein targets.
17. An evolving in meso screening strategy
As a community, we have close to two decades of experience with the in meso crystallization method. Have we learned any lessons that can be used for a more rational, less empirical approach to generating high-resolution structures with in meso-grown crystals? Certainly guidelines have emerged and several are presented below. Part of the problem with disseminating information of this type relates to the high-profile nature of many of the target proteins being reported. In consequence, much of the work is published in high-impact journals where space is at a premium and only the most essential experimental detail is included. The community suffers as a result by not being privy to the nature and extent of the screening performed that yielded the final structures. We have attempted to make good this deficit by reporting full details of the screening strategies implemented in the MS&FB Group with β-barrel and α-helical membrane-protein targets that have led to structures (Fig. 7; Li et al., 2011, 2014, 2015; Li, Shah et al., 2013). Some of the lessons learned from these assorted studies are summarized below in no particular order.
18. Facts and figures online
Further details regarding the structure and function of integral, anchored and peripheral membrane proteins are available online in a convenient and searchable database: the Membrane Protein Data Bank (MPDB; Raman et al., 2006; https://www.mpdb.tcd.ie ). Unfortunately, owing to a lack of resources, the database has not been updated since 2011. Whilst records in the MPDB include structure information directly from and hyperlinked to the PDB, they also contain additional useful data relating to the type of protein, the methods and materials used for and so on obtained from the source literature. Statistical analyses on the contents of the database, which, unlike the PDB, is limited to membrane proteins, can be performed and viewed conveniently online. Examples include `detergents used for membrane protein structure work', `number of structures published annually by method' and the like. Thus, while out of date, it still contains useful and searchable information. Perhaps, in time, it can be coupled to the PDB for automatic updating.
Over the years, the PDB has improved its search features. With more complete record annotation, hopefully to include full crystallization details, it may be that specialized databases such as the MPDB or that of Steve White's group (https://blanco.biomol.uci.edu/mpstruc/ ) will become redundant. This is as it should be, given the resources available to the PDB. As of this writing (September 2014), a `Search' of `Everything' from the PDB homepage (https://www.pdb.org ) under `lipidic cubic phase' yields 113 records and all are relevant. (Interestingly, a search under `lipid cubic phase' yields only 89 records.) However, by our reckoning, the PDB holds ∼192 such records (Figs. 1 and 2, Table 1). With this set of records, it is possible to create, at the press of the `Report' button, a very useful `customizable table' that can be sorted, filtered and eventually exported to an Excel file. It includes hyperlinks to PDB records and to most, but not all, annotated items in the standard PDB record. We look forward to enhanced functionality of this important, weekly updated resource.
19. Prospects
The in meso method burst onto the scene almost two decades ago (Fig. 2). It was received with great anticipation for what it would deliver; perhaps it was to be the panacea. However, output in the early years was limited to naturally abundant, bacterial α-helical proteins bedecked with stabilizing and highly coloured prosthetic groups. The perceived restricted range, coupled with the challenges associated with handling the sticky and viscous cubic meant that subsequent interest in the method waned. This was countered to some degree with the introduction of the in meso robot, a growing understanding of how the method worked at a molecular level and a continued demonstration of the general applicability of the method. However, interest in the method has rocketed of late with the success it has had, particularly in the GPCR field (Fig. 1, Table 1).
Improvements are needed for the method to thrive and for its longevity. Critically, the specialized materials and supplies upon which the method relies must be made more generally available and the method itself must be made more user-friendly and routine. New and improved in meso robots available on the market are tackling the user-friendliness issue. Workshops that involve hands-on demonstrations contribute to making the method more accessible. The author has been active in this area for past decade, with the latest workshop being held as part of the ICCBM15 meeting in Hamburg in September 2014 (https://www.iccbm15.org ). There, 72 students were trained in the practicalities and finer details of in meso crystallogenesis during the course of a three-day workshop (https://www.iccbm15.org/iccbm15_Workshop.xhtml ). Online, open-access video demonstrations of the method are available (Caffrey & Porter, 2010; Li, Boland, Aragão et al., 2012; Li, Boland, Walsh et al., 2012).
Developments are needed in the area of crystal identification. Optical clarity is of the highest quality with the glass sandwich plates currently in use and this provides the ready detection of colourless, micrometre-sized crystals in normal light and between crossed polarizers with a light microscope. Detection by UV fluorescence is particularly powerful and convenient for tryptophan-containing proteins. Fluorescence labelling (Forsythe et al., 2006) is also a route worth considering for the sensitive detection of early hits. Second-order nonlinear optical imaging of chiral crystals (SONICC) has been shown to sensitively and selectively detect certain membrane-protein crystals growing in meso (Kissick et al., 2010).
Recovering crystals from the ; Li, Boland, Aragão et al., 2012). This is especially true when harvesting is performed directly from glass sandwich plates. Typically, a glass cutter is used to open the well and to expose the Teasing out and harvesting the crystal for immediate diffraction or snap-cooling in liquid nitrogen is most conveniently performed with a cryo-loop. Harvesting is a slow, painstaking, inefficient and cumbersome process, especially if it must be performed in a cold room and/or in subdued light. This whole area of harvesting calls out for innovation.
for data collection is a nontrivial undertaking (Caffrey & Cherezov, 2009Data collection at a synchrotron is not exactly straightforward either. Given that in meso-grown crystals tend to be small, a micrometre-sized X-ray beam is required. Oftentimes, the crystal of interest is hidden from view in a bolus of opaque at 100 K on the cryo-loop. This means that locating the crystal and centring it requires rounds of diffraction rastering with a beam of progressively smaller size (Cherezov et al., 2009). This same approach is used to advantage in finding the best diffracting part of a crystal. Locating crystals and centring based on from heavy atoms in the sample is another option (Stepanov et al., 2011). Effective and efficient diffraction rastering is now recognized as an important feature of the most up-to-date MX beamlines at synchrotron facilities worldwide, and steady improvements in the rastering process are being made. In situ screening and data collection are other areas that are under vigorous investigation (Bingel-Erlenmeyer et al., 2011; Axford et al., 2012). The in situ approach will benefit from improvements in sample presentation, high-performance goniometers, higher X-ray fluence, smaller and more stable beams, and faster detectors. Also, in the interests of the environment, cost, time and convenience, remote screening and data collection that is as easy and as efficient as it is on-site must be implemented.
Seeding has been used to advantage, especially with soluble proteins, to enable the production of structure-grade crystals in recalcitrant systems. Indeed, the recently introduced matrix seeding is proving to be particularly successful (D'Arcy et al., 2014). A seeding protocol that is applicable to in meso crystallization would certainly be well received. Issues that need to be resolved include establishing that seeding actually works in meso and, if it does, the type of seed crystals and conditions that are most effective. Must seed crystals be grown in meso or can they be provided from alternative crystallization sources? It may be that seed crystals generated in meso could be used for crystallization trials in surfo. We have established that the bilayer of the cubic phase is fusogenic (Caffrey, 2008, 2009). Therefore, combining two boluses of one with seed crystals and the other with target protein reconstituted under conditions that place it in the so-called nucleation zone, should in principle provide the conditions for seed-induced crystal growth. This area is deserving of further study.
Without exception, all 192 records in the PDB that refer to in meso structures exhibit layered or type I crystal packing. Whilst alternative packing arrangements are theoretically possible (§2), for the moment a very reasonable assumption is that type I packing prevails. If so, then it occurs to the author that this layered packing information might be used to advantage to solve in meso crystal structures. However, demonstrating that this is in fact useful `prior knowledge' and that it can be exploited must be left to a suitably skilled and motivated crystallographer.
Given that membrane proteins are important drug targets, obtaining high-resolution crystal structures of target proteins with ligands bound is an important goal. Ligands often have limited water solubility. The bilayer of the et al., 2015). This same approach is also worth exploring with water-soluble proteins that we know can be crystallized in meso and with poorly soluble ligands made available at high concentrations by way of a surrounding, very local bilayer.
can be used to advantage here as a reservoir from which ligands are provided by way of the bilayer itself or the aqueous compartments of the (see LiFinally, the method should begin to be used with really large proteins and complexes. The sponge phase (Cherezov, Clogston et al., 2006), with its open aqueous channels and flatter, less rigid bilayer, should prove particularly useful in this regard. Using it in combination with novel hosting and additive lipid screens (Cherezov, Clogston et al., 2006; Li et al., 2011, 2014; Li, Shah et al., 2013) will go a long way towards producing crystals and ultimately high-resolution structures where interactions that are integral to human health are revealed.
Acknowledgements
There are many who have contributed to this work. Most are from my own group, both past and present members. To all I extend my warmest thanks and appreciation. This work was supported, in part, by grants from Science Foundation Ireland (12/IA/1255) and the National Institutes of Health (GM75915, P50GM073210 and U54GM094599).
References
Aherne, M., Lyons, J. A. & Caffrey, M. (2012). A fast, simple and robust protocol for growing crystals in the lipidic cubic phase. J. Appl. Cryst. 45, 1330–1333. Web of Science CrossRef CAS IUCr Journals Google Scholar
Ai, X. & Caffrey, M. (2000). Membrane protein crystallization in lipidic mesophases: detergent effects. Biophys. J. 79, 394–405. Google Scholar
Axford, D. et al. (2012). In situ macromolecular crystallography using microbeams. Acta Cryst. D68, 592–600. Web of Science CrossRef CAS IUCr Journals Google Scholar
Barends, T. R., Foucar, L., Botha, S., Doak, R. B., Shoeman, R. L., Nass, K., Koglin, J. E., Williams, G. J., Boutet, S., Messerschmidt, M. & Schlichting, I. (2014). De novo protein crystal structure determination from X-ray free-electron laser data. Nature (London), 505, 244–247. CAS Google Scholar
Berman, H. M., Henrick, K. & Nakamura, H. (2003). Announcing the Worldwide Protein Data Bank (2003). Nature Struct. Biol. 10, 1203–980. Google Scholar
Bingel-Erlenmeyer, R., Olieric, V., Grimshaw, J. P. A., Gabadinho, J., Wang, X., Ebner, S. G. & Schulze-Briese, C. (2011). SLS crystallization platform at beamline X06DA – a fully automated pipeline enabling in situ X-ray diffraction screening. Cryst. Growth Des. 11, 916–923. CAS Google Scholar
Boland, C., Li, D., Shah, S. T. A., Haberstock, S., Dötsch, V., Bernhard, F. & Caffrey, M. (2014). Cell-free expression and in meso crystallisation of an integral membrane kinase for structure determination. Cell. Mol. Life Sci. 71, 4895–4910. CAS Google Scholar
Briggs, J. & Caffrey, M. (1994). The temperature-composition phase diagram of monomyristolein in water: equilibrium and metastability aspects. Biophys. J. 66, 573–587. Google Scholar
Burmeister, W. P. (2000). Structural changes in a cryo-cooled protein crystal owing to radiation damage. Acta Cryst. D56, 328–341. CrossRef IUCr Journals Google Scholar
Caffrey, M. (1987). Kinetics and mechanism of transitions involving the lamellar, cubic, inverted hexagonal and fluid isotropic phase of hydrated monoacylglycerides monitored by time-resolved X-ray diffraction. Biochemistry, 26, 6349–6363. Google Scholar
Caffrey, M. (2000). A lipid's eye view of membrane protein crystallization in mesophases. Curr. Opin. Struct. Biol. 10, 486–497. CAS Google Scholar
Caffrey, M. (2003). Membrane protein crystallization. J. Struct. Biol. 142, 108–132. CAS Google Scholar
Caffrey, M. (2008). On the mechanism of membrane protein crystallization in lipidic mesophases. Cryst. Growth Des. 8, 4244–4254. CAS Google Scholar
Caffrey, M. (2009). Crystallizing membrane proteins for structure determination. Use of lipidic mesophases. Annu. Rev. Biophys. 38, 29–51. CAS Google Scholar
Caffrey, M. (2011). Crystallizing membrane proteins for structure–function studies using lipidic mesophases. Biochem. Soc. Trans. 39, 725–732. CAS Google Scholar
Caffrey, M. (2013). Crystallizing membrane proteins for structure–function studies using lipidic mesophases. In Advancing Methods for Biomolecular Crystallography, edited by R. J. Read, A. G. Urzhumtsev & V. Y. Lunin, pp. 33–46. Dordrecht: Springer. Google Scholar
Caffrey, M. & Cherezov, V. (2009). Crystallizing membrane proteins in lipidic mesophases. Nature Protoc. 4, 706–731. CAS Google Scholar
Caffrey, M., Eifert, R., Li, D. & Howe, N. (2014). The lipid cubic phase or in meso method for crystallizing proteins. Bushings for better manual dispensing. J. Appl. Cryst. 47, 1804–1806. CrossRef CAS PubMed IUCr Journals Google Scholar
Caffrey, M., Li, D. & Dukkipati, A. (2012). Membrane protein structure determination using crystallography and lipidic mesophases: recent advances and successes. Biochemistry, 51, 6266–6288. Google Scholar
Caffrey, M., Li, D., Howe, N. & Syed, S. T. A. (2014). `Hit and run' serial femtosecond crystallography of a membrane kinase in the lipid cubic phase. Philos. Trans. R. Soc. Lond. B Biol. Sci. 369, 20130621. CrossRef Google Scholar
Caffrey, M., Lyons, J., Smyth, T. & Hart, D. J. (2009). Monoacylglycerols. The workhorse lipids for crystallizing membrane proteins in mesophases. Curr. Top. Membr. 63, 83–108. CAS Google Scholar
Caffrey, M. & Porter, C. (2010). Crystallizing membrane proteins for structure determination using lipidic mesophases. J. Vis. Exp. 45, e1712. Google Scholar
Chapman, H. N., Caleman, C. & Timneanu, N. (2014). Diffraction before destruction. Philos. Trans. R. Soc. Lond. B Biol. Sci. 369, 20130313. CrossRef Google Scholar
Chapman, H. N. et al. (2011). Femtosecond X-ray protein nanocrystallography. Nature (London), 470, 73–77. CAS Google Scholar
Chen, Y.-J., Pornillos, O., Lieu, S., Ma, C., Chen, A. P. & Chang. G. (2007). X-ray structure of EmrE supports dual topology model. Proc. Natl Acad. Sci. USA, 104, 18999–19004. Google Scholar
Cheng, A., Hummel, B., Qiu, H. & Caffrey, M. (1998). A simple mechanical mixer for small viscous lipid-containing samples. Chem. Phys. Lipids, 95, 11–21. CAS Google Scholar
Cherezov, V. & Caffrey, M. (2003). Nano-volume plates with excellent optical properties for fast, inexpensive crystallization screening of membrane proteins. J. Appl. Cryst. 36, 1372–1377. Google Scholar
Cherezov, V. & Caffrey, M. (2005). A simple and inexpensive nanolitre-volume dispenser for highly viscous materials used in membrane protein crystallization. J. Appl. Cryst. 38, 398–400. CrossRef CAS IUCr Journals Google Scholar
Cherezov, V. & Caffrey, M. (2006). Picolitre-scale crystallization of membrane proteins. J. Appl. Cryst. 39, 604–606. CrossRef CAS IUCr Journals Google Scholar
Cherezov, V. & Caffrey, M. (2007). Membrane protein crystallization in lipidic mesophases. A mechanism study using X-ray microdiffraction. Faraday Discuss. 136, 195–212. CAS Google Scholar
Cherezov, V., Clogston, J., Misquitta, Y., Abdel-Gawad, W. & Caffrey, M. (2002). Membrane protein crystallization in meso: lipid type-tailoring of the cubic phase. Biophys. J. 83, 3393–3407. Google Scholar
Cherezov, V., Clogston, J., Papiz, M. & Caffrey, M. (2006). Room to move. Crystallizing membrane proteins in swollen lipidic mesophases. J. Mol. Biol. 357, 1605–1618. CAS Google Scholar
Cherezov, V., Fersi, H. & Caffrey, M. (2001). Crystallization screens: compatibility with the lipidic cubic phase for in meso crystallization of membrane proteins. Biophys. J. 81, 225–242. Google Scholar
Cherezov, V., Hanson, M. A., Griffith, M. T., Hilgart, M. C., Sanishvili, R., Nagarajan, V., Stepanov, S., Fischetti, R. F., Kuhn, P. & Stevens, R. C. (2009). Rastering strategy for screening and centring of microcrystal samples of human membrane proteins with a sub-10 µm size X-ray synchrotron beam. J. R. Soc. Interface, 6, S587–S597. CAS Google Scholar
Cherezov, V., Liu, W., Derrick, J., Luan, B., Aksimentiev, A., Katrich, V. & Caffrey, M. (2008). In meso crystal structure and docking simulations suggest an alternative proteoglycan binding site in the OpcA outer membrane adhesin. Proteins, 71, 24–34. CAS Google Scholar
Cherezov, V., Peddi, A., Muthusubramaniam, L., Zheng, Y. F. & Caffrey, M. (2004). A robotic system for crystallizing membrane and soluble proteins in lipidic mesophases. Acta Cryst. D60, 1795–1807. CrossRef CAS IUCr Journals Google Scholar
Cherezov, V., Rosenbaum, D. M., Hanson, M. A., Rasmussen, S. G. F., Thian, F. S., Kobilka, T. S., Choi, H.-J., Kuhn, P., Weis, W. I., Kobilka, B. K. & Stevens, R. C. (2007). High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor. Science, 318, 1258–1265. CAS Google Scholar
Cherezov, V., Yamashita, E., Liu, W., Zhalnina, M., Cramer, W. A. & Caffrey, M. (2006). In meso structure of the cobalamin transporter, BtuB, at 1.95 Å resolution. J. Mol. Biol. 364, 716–734. Google Scholar
Chung, H. & Caffrey, M. (1994). The curvature elastic energy function of the lipid–water cubic mesophase. Nature (London), 368, 224–226. CAS Google Scholar
Clogston, J. & Caffrey, M. (2005). Controlling release from the cubic phase. Amino acids, peptides, proteins and nucleic acids. J. Control. Release, 107, 97–111. Google Scholar
Clogston, J., Graciun, G., Hart, D. J. & Caffrey, M. (2005). Controlling release from the lipidic cubic phase by selective alkylation. J. Control. Release, 102, 441–461. CAS Google Scholar
Darmanin, C., Conn, C. E., Newman, J., Mulet, X., Seabrook, S. A., Liang, Y.-L., Hawley, A., Kirby, N., Varghese, J. N. & Drummond, C. J. (2012). High-throughput production and structural characterization of libraries of self-assembly lipidic cubic phase materials. ACS Comb. Sci. 14, 247–252. CAS Google Scholar
D'Arcy, A., Bergfors, T., Cowan-Jacob, S. W. & Marsh, M. (2014). Microseed matrix screening for optimization in protein crystallization: what have we learned? Acta Cryst. F70, 1117–1126. Google Scholar
Fairman, J. W., Dautin, N., Wojtowicz, D., Liu, W., Noinaj, N., Barnard, T. J., Udho, E., Przytycka, T. M., Cherezov, V. & Buchanan, S. K. (2012). Crystal structures of the outer membrane domain of intimin and invasin from enterohemorrhagic E. coli and enteropathogenic Y. pseudotuberculosis. Structure, 20, 1233–1243. CAS Google Scholar
Forsythe, E., Achari, A. & Pusey, M. L. (2006). Trace fluorescent labeling for high-throughput crystallography. Acta Cryst. D62, 339–346. Web of Science CrossRef CAS IUCr Journals Google Scholar
Fraser, J. S., van den Bedem, H., Samelson, A. J., Lang, P. T., Holton, J. M., Echols, N. & Alber, T. (2011). Accessing protein conformational ensembles using room-temperature X-ray crystallography. Proc. Natl Acad. Sci. USA, 108, 16, 247–16252. Google Scholar
Grabe, M. Neu, J., Oster, G. & Nollert, P. (2003). Protein interactions and membrane geometry. Biophys. J. 84, 854–868. CAS Google Scholar
Hato, M., Minamikawa, H., Salkar, R. A. & Matsutani, S. (2004). Phase behavior of phytanyl-chained akylglycoside/water systems. Prog. Colloid Polym. Sci. 123, 56–60. CAS Google Scholar
Höfer, N., Aragão, D. & Caffrey, M. (2010). Crystallizing transmembrane peptides in lipidic mesophases. Biophys. J. 99, L23–L25. Google Scholar
Höfer, N., Aragão, D. & Caffrey, M. (2011). The lipidic cubic phase as a membrane mimetic. Biophys. J. 100, 2075. Google Scholar
Höfer, N., Aragão, D., Lyons, J. M. & Caffrey, M. (2011). Membrane protein crystallization in lipidic mesophases. Hosting lipid effects on the crystallization and structure of a transmembrane peptide. Cryst. Growth Des. 11, 1182–1192. Google Scholar
Johansson, L. C. et al. (2012). Lipidic phase membrane protein serial femtosecond crystallography. Nature Methods, 9, 263–265. CAS Google Scholar
Johner, N., Mondal, S., Morra, G., Caffrey, M., Weinstein, H. & Khelashvili, G. (2014). Protein and lipid interactions driving molecular mechanisms of in meso crystallization. J. Am. Chem. Soc. 136, 3271–3284. CAS Google Scholar
Kato, H. E., Zhang, F., Yizhar, O., Ramakrishnan, C., Nishizawa, T., Hirata, K., Ito, J., Aita, Y., Tsukazaki, T., Hayashi, S., Hegemann, P., Maturana, A. D., Ishitani, R., Deisseroth, K. & Nureki, O. (2012). Crystal structure of the channelrhodopsin light-gated cation channel. Nature (London), 482, 369–374. CAS Google Scholar
Khelashvili, G., Albornoz, P., Johner, N., Mondal, S., Caffrey, M. & Weinstein, H. (2012). Why GPCRs behave differently in cubic and lamellar lipidic mesophases. J. Am. Chem. Soc. 134, 15858–15868. CAS Google Scholar
Kissick, D. J., Gualtieri, E. J., Simpson, G. J. & Cherezov, V. (2010). Nonlinear optical imaging of integral membrane proteins in lipidic mesophases. Anal. Chem. 82, 491–497. CAS Google Scholar
Landau, E. M., Rummel, G., Cowan-Jacob, S. W. & Rosenbusch, J. P. (1997). Crystallization of a polar protein and small molecules from the aqueous compartment of lipidic cubic phases. J. Phys. Chem. B, 101, 1935–1937. CAS Google Scholar
Li, D., Boland, C., Aragão, D., Walsh, K. & Caffrey, M. (2012). Harvesting and cryo-cooling crystals of membrane proteins grown in lipidic mesophases for structure determination by macromolecular crystallography. J. Vis. Exp. 67, e4001. Google Scholar
Li, D., Boland, C., Walsh, K. & Caffrey, M. (2012). Use of a robot for high-throughput crystallization of membrane proteins in lipidic mesophase. J. Vis. Exp. 67, e4000. Google Scholar
Li, D. & Caffrey, M. (2011). The lipidic cubic phase as a membrane mimetic for integral membrane protein enzymes. Proc. Natl Acad. Sci. USA, 108, 8639–8644. CAS Google Scholar
Li, D. & Caffrey, M. (2014). Renaturing membrane proteins in the lipid cubic phase, a nanoporous membrane mimetic. Sci. Rep. 4, 5806. Google Scholar
Li, D. et al. (2014). Crystallizing membrane proteins in the lipidic mesophase. Experience with human prostaglandin E2 synthase 1 and an evolving strategy. Cryst. Growth Des. 14, 2034–2047. CAS Google Scholar
Li, D., Lee, J. & Caffrey, M. (2011). Crystallizing membrane proteins in lipidic mesophases. A host lipid screen. Cryst. Growth Des. 11, 530–537. CAS Google Scholar
Li, D., Lyons, J. A., Pye, V. E., Vogeley, L., Aragão, D., Kenyon, C. P., Shah, S. T. A., Doherty, C., Aherne, M. & Caffrey, M. (2013). Crystal structure of the integral membrane diacylglycerol kinase. Nature (London), 497, 521–524. CAS Google Scholar
Li, D., Pye, V. E. & Caffrey, M. (2015). Experimental phasing for structure determination using membrane protein crystals grown by the lipid cubic phase method. Acta Cryst. D71, doi:10.1107/S1399004714010360. Google Scholar
Li, D., Shah, S. T. A. & Caffrey, M. (2013). Host lipid and temperature as important screening variables for crystallizing integral membrane proteins in lipidic mesophases. Trials with diacylglycerol kinase. Cryst. Growth Des. 13, 2846–2857. CAS Google Scholar
Liao, J., Li, H., Zeng, W., Sauer, D. B., Belmares, R. & Jiang, Y. (2012). Structural insight into the ion-exchange mechanism of the sodium/calcium exchanger. Science, 335, 686–690. CAS Google Scholar
Liu, W. & Caffrey, M. (2005). Gramicidin structure and disposition in highly curved membranes. J. Struct. Biol. 150, 23–40. CAS Google Scholar
Liu, W. & Caffrey, M. (2006). Interactions of tryptophan, tryptophan peptides and tryptophan alkyl esters at curved membrane interfaces. Biochemistry, 45, 11713–11726. Google Scholar
Liu, W., Ishchenko, A. & Cherezov, V. (2014). Preparation of microcrystals in lipidic cubic phase for serial femtosecond crystallography. Nature Protoc. 9, 2123–2134. CAS Google Scholar
Liu, W. et al. (2013). Serial femtosecond crystallography of G protein-coupled receptors. Science, 342, 1521–1524. CAS Google Scholar
Lyons, J. A., Aragão, D., Slattery, O., Pisliakov, A. V., Soulimane, T. & Caffrey, M. (2012). Structural insights into electron transfer in caa3-type cytochrome oxidase. Nature (London), 487, 514–518. CAS Google Scholar
Lyons, J. A., Parker, J. L., Solcan, N., Brinth, A., Li, D., Shah, S. T. A., Caffrey, M. & Newstead, S. (2014). Structural basis for polyspecificity in the POT family of proton-coupled oligopeptide transporters. EMBO Rep. 15, 886–893. CAS Google Scholar
Malinauskaite, L., Quick, M., Reinhard, L., Lyons, J. A., Yano, H., Javitch, J. A. & Nissen, P. (2014). A mechanism for intracellular release of Na by neurotransmitter: sodium symporters. Nature Mol. Struct. Biol. 21, 1006–1012. Google Scholar
Misquitta, Y. & Caffrey, M. (2003). Detergents destabilize the cubic phase of monoolein: implications for membrane protein crystallization. Biophys. J. 85, 3084–3096. Google Scholar
Misquitta, Y., Cherezov, V., Havas, F., Patterson, S., Mohan, J., Wells, A. J., Hart, D. J. & Caffrey, M. (2004). Rational design of lipid for membrane protein crystallization. J. Struct. Biol. 148, 169–175. CAS Google Scholar
Misquitta, L. V., Misquitta, Y., Cherezov, V., Slattery, O. & Mohan, J. M. (2004). Membrane protein crystallization in lipidic mesophases with tailored bilayers. Structure, 12, 2113–2124. CAS Google Scholar
Qiu, H. & Caffrey, M. (2000). Phase diagram of the monoolein/water system: metastability and equilibrium aspects. Biomaterials, 21, 223–234. Google Scholar
Raman, P., Cherezov, V. & Caffrey, M. (2006). The Membrane Protein Data Bank. Cell. Mol. Life Sci. 63, 36–51. Google Scholar
Rasmussen, S. G. F., Choi, H.-J. et al. (2011). Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature (London), 469, 175–180. CAS Google Scholar
Rasmussen, S. G. F., DeVree, B. T. et al. (2011). Crystal structure of the β2 adrenergic receptor–Gs protein complex. Nature (London), 477, 549–555. CAS Google Scholar
Ring, A. M., Manglik, A., Kruse, A. C., Enos, M. D., Weis, W. I., Garcia, K. C. & Kobilka, B. K. (2013). Adrenaline-activated structure of β2-adrenoceptor stabilized by an engineered nanobody. Nature (London), 502, 575–579. CAS Google Scholar
Schwarz, D., Junge, F., Durst, F., Frolich, N., Schneider, B., Reckel, S., Sobhanifar, S., Dötsch, V. & Bernhard, F. (2007). Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems. Nature Protoc. 2, 2945–2957. CAS Google Scholar
Sciara, G., Clarke, O. B., Tomasek, D., Kloss, B., Tabuso, S., Byfield, R., Cohn, R., Banerjee, S., Rajashankar, K. R., Slavkovic, V., Graziano, J. H., Shapiro, L. & Mancia, F. (2014). Structural basis for catalysis in a CDP-alcohol phosphotransferase. Nature Commun. 5, 4068. Google Scholar
Spence, J. C. H., Weierstall, U. & Chapman, H. N. (2012). X-ray lasers for structural and dynamic biology. Rep. Prog. Phys. 75, 102601. Google Scholar
Stepanov, S., Hilgart, M., Yoder, D. W., Makarov, O., Becker, M., Sanishvili, R., Ogata, C. M., Venugopalan, N., Aragão, D., Caffrey, M., Smith, J. L. & Fischetti, R. F. (2011). Fast fluorescence techniques for crystallography beamlines. J. Appl. Cryst. 44, 772–778. CrossRef CAS IUCr Journals Google Scholar
Suzuki, H., Nishizawa, T., Tani, K., Yamazaki, Y., Tamura, A., Ishitani, R., Dohmae, N., Tsukita, S., Nureki, O. & Fujiyoshi, Y. (2014). Crystal structure of a claudin provides insight into the architecture of tight junctions. Science, 344, 304–307. CAS Google Scholar
Takeda, H., Hattori, M., Nishizawa, T., Yamashita, K., Shah, S. T. A., Caffrey, M., Maturana, A. D., Ishitani, R. & Nureki, O. (2014). Structural basis for ion selectivity revealed by high-resolution crystal structure of Mg2+ channel MgtE. Nature Commun. 5, 5374. Google Scholar
Tanaka, S., Egelhaaf, S. U. & Poon, W. C. K. (2004). Crystallization of a globular protein in lipid cubic phase. Phys. Rev. Lett. 92, 128102. Google Scholar
Wada, T., Shimono, K., Kikukawa, T., Hato, M., Shinya, N., Kim, S. Y., Kimura-Someya, T., Shirouzu, M., Tamogami, J., Miyauchi, S., Jung, K.-H., Kamo, N. & Yokoyama, S. (2011). Crystal structure of the eukaryotic light-driven proton-pumping rhodopsin, Acetabularia rhodopsin II, from marine alga. J. Mol. Biol. 411, 986–998. Google Scholar
Weierstall, U. et al. (2014). Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography. Nature Commun. 5, 3309. Google Scholar
Weinert, T. et al. (2014). Fast native-SAD phasing for routine macromolecular structure determination. Nature Methods, doi:10.1038/nmeth.3211. Google Scholar
Wöhri, A. B., Johansson, L. C., Wadsten-Hindrichsen, P., Wahlgren, W. Y., Fischer, G., Horsefield, R., Katona, G., Nyblom, M., Öberg, F., Young, G., Cogdell, R. J., Fraser, N. J., Engström, S. & Neutze, R. (2008). A lipidic sponge phase screen for membrane protein crystallization. Structure, 16, 1003–1009. Google Scholar
Yang, D., Cwynar, V. A., Hart, D. J., Madanmohan, J., Lee, J., Lyons, J. & Caffrey, M. (2012). Preparation of 1-monoacylglycerols via the Suzuki–Miyaura reaction: 2,3-dihydroxypropyl (Z)-tetradec-7-enoate. Org. Synth. 89, 183–201. Google Scholar
This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.