The cubicon method for concentrating membrane proteins in ... · nature protocols || VOL.12 NO.9 2017 | 1745 IntroDuctIon The primary application of the lipid cubic phase or in meso

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© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

protocol

nature protocols | VOL.12 NO.9 | 2017 | 1745

IntroDuctIonThe primary application of the lipid cubic phase or in meso method is to crystallize membrane proteins1–3. The crystals are used for macromolecular X-ray crystallography, leading to high-resolution structures that provide insight into the mechanism of action of the protein. For medically relevant protein targets, struc-tures are used to inform drug discovery4,5. The in meso method is experiencing explosive growth as more groups embrace and suc-ceed with it3. Currently, there are 392 records in the Protein Data Bank (PDB) attributed to the method, representing 126 unique structures and 18 family classes (Fig. 1a). Of particular note is the fact that nearly 50% of these records were entered into the PDB in the past 2 years. The in meso method works by reconstitut-ing the protein into a bicontinuous, native-like lipid bilayer that facilitates nucleation and growth of membrane protein crystals. The method has been the subject of a previous Protocol1. Here, we describe a general methodology with which to concentrate membrane proteins in the cubic mesophase by sequentially reconstituting a dilute solution of the solubilized protein into the cubic mesophase to ramp up the protein concentration to values suitable for in meso crystallization.

A challenge associated with the in meso methodAn important challenge faced by the in meso method—one it shares with other crystallization techniques—is the need for relatively high starting protein concentrations (Fig. 1). The more supersaturated the system, the greater is the driving force for nucleation leading to crystal growth. Ideally, the goal is to favor nucleation first, by working at the highest possible protein concentration. Once nuclei form, the protein concentration can

be reduced to just above the (super)solubility limit. Under these conditions the slow, orderly growth of a few, good-quality crystals can occur. These same principles are likely to apply to in meso crystallization, where, to begin with, the highest possible protein concentration should be used to support nucleation. However, some proteins simply cannot be concentrated to the levels required for crystallization. In the process of being concentrated, the protein aggregates and falls out of solution.

A solution to this very practical and limiting problem is described here. This protocol involves sequential rounds of reconstitution, during which the protein concentration in the bilayer of the cubic phase rises incrementally with each round6. This ‘cubicon’ method benefits from the finite water (aqueous solution)-carrying capacity of the mesophase, beyond which the system phase separates7, as well as from the natural tendency of a membrane protein to partition from an aqueous solution into a mesophase bilayer. Thus, by repeating the reconstitu-tion step with a single mesophase bolus and a series of dilute protein solutions, the protein load in the bilayer of the meso-phase increases with each round, leaving excess, phase-separated aqueous solution depleted of protein. This protein-depleted solution is removed between rounds of reconstitution. The final, protein-enriched mesophase can then be used in crystalliza-tion trials. In the interest of completeness, alternative methods that have been used to concentrate proteins are summarized in Table 1.

The cubicon method works for two reasons. First, membrane proteins are amphiphilic and will naturally preferentially parti-tion from an aqueous solution into the bilayer of the cubic phase

The cubicon method for concentrating membrane proteins in the cubic mesophasePikyee Ma1,5, Dietmar Weichert1,5 , Luba A Aleksandrov2, Timothy J Jensen2, John R Riordan2, Xiangyu Liu3, Brian K Kobilka4 & Martin Caffrey1

1Membrane Structural and Functional Biology Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland. 2Department of Biochemistry and Biophysics and Cystic Fibrosis Treatment and Research Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. 3Beijing Advanced Innovation Center for Structural Biology, School of Medicine, Tsinghua University, Beijing, China. 4Department of Molecular and Cellular Physiology, School of Medicine, Stanford University, Stanford, California, USA. 5These authors contributed equally to this work. Correspondence should be addressed to M.C. ([email protected]).

Published online 3 August 2017; doi:10.1038/nprot.2017.057

the lipid cubic phase (in meso) method is an important approach for generating crystals and high-resolution X-ray structures of integral membrane proteins. However, as a consequence of instability, it can be impossible—using traditional methods—to concentrate certain membrane proteins and complexes to values suitable for in meso crystallization and structure determination. the cubicon method described here exploits the amphiphilic nature of membrane proteins and their natural tendency to partition preferentially into lipid bilayers from aqueous solution. using several rounds of reconstitution, the protein concentration in the bilayer of the cubic mesophase can be ramped up stepwise from less than a milligram per milliliter to tens of milligrams per milliliter for crystallogenesis. the general applicability of the method is demonstrated with five integral membrane proteins: the b2-adrenergic G protein-coupled receptor (b2ar), the peptide transporter (peptst), diacylglycerol kinase (Dgka), the alginate transporter (alge) and the cystic fibrosis transmembrane conductance regulator (cFtr). In the cases of b2ar, peptst, Dgka and alge, an effective 20- to 45-fold concentration was realized, resulting in a protein-laden mesophase that allowed the formation of crystals using the in meso method and structure determination to resolutions ranging from 2.4 Å to 3.2 Å. In addition to opening up in meso crystallization to a broader range of integral membrane protein targets, the cubicon method should find application in situations that require membrane protein reconstitution in a lipid bilayer at high concentrations. these applications include functional and biophysical characterization studies for ligand screening, drug delivery, antibody production and protein complex formation. a typical cubicon experiment can be completed in 3–5 h.

http://orcid.org/0000-0002-0694-7671

http://dx.doi.org/10.1038/nprot.2017.057

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1746 | VOL.12 NO.9 | 2017 | nature protocols

with which the solution is homogenized. Second, according to the corresponding temperature versus composition phase diagram for the lipid that creates the hosting mesophase3,7, the mesophase is stable in equilibrium with an excess of aqueous solution. This means that the mesophase can be separated physically from the aqueous solution after it has given up its protein manifest to the mesophase. Fortunately, the mesophase is viscous and tends to stick to itself. This facilitates separation of the protein-depleted aqueous solution from the protein-laden mesophase in bulk for use in crystallogenesis.

Development of the protocolMotivation for developing the cubicon method came, in part, from ongoing efforts to obtain high-resolution crystal structures of the human CFTR8. CFTR is a chloride channel in the api-cal membrane of epithelial cells. Mutant forms of the protein give rise to cystic fibrosis, a genetic disease leading to premature death. A crystal structure is sought with a view to understanding the mechanism of action and regulation, and for structure-based drug design. However, in certain detergent solutions the protein is refractory to a concentration above ~0.5 mg/ml. This has limited its use in crystallographic studies9. As reported here, the cubicon method has enabled CFTR to be concentrated to the equivalent of 18 mg/ml, a value well within the range considered suitable for in meso crystallization and structure determination (Fig. 1).

CFTR is expensive to produce and is available in relatively small amounts. Thus, the development and exploratory work upon which the cubicon method is based was done with four structur-ally diverse reference proteins, all of which have been crystal-lized by the in meso method yielding structures. These include the α-helical diacylglycerol kinase (DgkA)10, the β2 adrenergic G protein-coupled receptor (β2AR)11, the peptide transporter (PepTSt)12 and the β-barrel alginate transporter (AlgE)13. By

implementing the cubicon method, 10–17 rounds of reconstitution were used to raise protein levels by 20- to 45-fold to effective values of 10–45 mg/ml starting protein concentration. In all four cases, the protein-enriched mesophase was used to generate crystals by the in meso method and structures with resolutions ranging from 2.4 Å to 3.2 Å. Crystallization trials with CFTR are ongoing. The cubicon method should find general applicability in the membrane structural and functional biology field.

The idea of using rounds of reconstitution to incrementally raise the concentration of protein in the bilayer of the lipid cubic phase as a prelude to in meso crystallogenesis was first mooted over a decade ago14. We now demonstrate that the cubicon method is generally applicable, proving effective with a variety of structu-rally diverse α-helical and β-barrel integral membrane proteins of sizes ranging from 15 to 180 kDa. It is compatible with pro-teins that exist as monomers, multimers and as ligand complexes. Further, it works with common detergents (dodecyl β-d-malto-side (DDM), the maltose neopentyl glycol (MNG) series, lauryl dimethylamine-N-oxide (LDAO)) and hosting lipids (9.9 monoa-cylglycerol (MAG), 9.7 MAG) alone and in combination with additive lipids (cholesterol). In addition, it can generate meso-phases covering a wide range of final protein concentrations.

A major concern that arose in the course of developing the cubicon method was the rising detergent concentration in the mesophase that would accompany the rounds of reconstitution performed with a dilute protein–detergent solution. The possibil-ity existed that the detergent load would overwhelm the system, completely destabilizing the cubic phase in favor of the lamellar phase, which alone is not considered compatible with in meso crystallogenesis15. Here, we demonstrate that the cubic phase has an impressive carrying capacity for detergent before it destabilizes (Fig. 2). That capacity is many fold in excess of the maximum amount of detergent that could possibly be incorporated into

Adhesin (3)10–40 mg/ml

Insertase (3)6–15 mg/ml

Porin (3)40–50 mg/ml

Hormone-bindingreceptor (2)15 mg/ml

Junction protein (1)7 mg/ml

Light-harvestingcomplex (1)12 mg/ml

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140

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60

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20

Protein concentration (mg/ml)

No.

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<100

Pore-formingtoxins (1)10 mg/ml

Lipoprotein signalpeptidase (1)

12 mg/ml

Cell invasion (1)30–40 mg/ml

GPCR (110)12–100 mg/ml

Rhodopsin (95)9–60 mg/ml

Transporter (65)8–50 mg/ml

Single-passreceptor (2)

40–100 mg/ml

Ion channel (5)12–45 mg/ml

Photosyntheticreaction centre (14)

6–35 mg/ml

Cytochromeoxidase (15)10–30 mg/ml

Exchanger (18)40–50 mg/ml

Lipid metabolismenzyme (31)10–60 mg/ml

a b

Figure 1 | Membrane protein types yielding structures based on in meso crystallization and the corresponding protein concentrations used for crystallization. (a) Pie chart showing the number of crystal structures (in parentheses) per protein family type solved using the in meso method. The protein concentration range of the solutions used for crystallization is listed below the family name. (b) Frequency histogram of membrane protein structures solved versus protein concentration used to set up crystallization trials. Records are included that refer to mesophase preparation by mixing aqueous protein solution with lipid. Records referring to other mesophase preparation methods, such as those used with the peptide gramicidin, are not included. Data gathered from the PDB on 9 March 2017.

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the mesophase in the multiple rounds of reconstitution used in this study. These findings engender confidence in the cubicon method and its more general applicability in that commonly used detergents at concentrations encountered in typical applications should not create problems with mesophase instability during its implementation.

The cubicon method involves multiple rounds of reconstitu-tion during which the detergent load in the mesophase increases in parallel with the desired rising protein concentration. High concentrations of detergents destabilize the cubic phase in which crystallogenesis occurs and should be avoided15–17. Tolerance for detergent depends not only on surfactant concentration but also

table 1 | Alternative methods for concentrating membrane proteins.

Method pros cons

concentrators: A separator with a porous membrane of defined molecular-weight (MW) cutoff size can be used to concentrate a solution of protein by filtration. The filtration process is facilitated by centrifugation, positive pressure (typically N2 gas) or by applying vacuum

• Simple to implement, no complex or expensive equipment needed

• No additives are used; the sample is not contaminated

• A range of MW cutoffs are available to suit different applications

• Fast • Adaptable to high-throughput applications • Commercially available • Extensively used, with a good track record • Protein can be concentrated to defined

levels—within limits • The fold concentration that is possible ranges

from low to high • Minimal protein loss, good protein recovery

• Concentrators show quite variable performance depending on the manufacturer

• Protein can stick to and block the membrane

• Membranes leak occasionally • Protein may be unstable and precipitate

during the concentration process • Depending on MW cutoff size, detergent

micelles and other high-MW solutes and aggregates may concentrate along with the protein

• Retentate viscosity and handling issues can arise at high concentrations of protein and detergent

• The minimum volume of starting solution needed varies with concentrator type

affinity chromatography: The protein is concentrated by binding it specifically and tightly to a ligand immobilized on a minimal volume of chromato-graphic resin, followed by elution with free ligand or by adjusting pH in a minimal volume of solution

• Can be an integral part of the purification process

• Fast • Adaptable to high-throughput applications • Commercially available • Extensively used, with a good track record

• Specific ligands may be required • Protein may need to be affinity tagged

(e.g., His-tag, GST) • The protein fold concentration that is

possible is limited • Sample is contaminated with eluting

compound

precipitation: Phase-separation of the protein from solvent as aggregated or precipitated material upon the addition of denaturants such as salt (e.g., ammonium sulfate), trichloroacetic acid, solvents (e.g., ethanol, acetone) and polymers (e.g., PEG)

• A simple, fast process • No complex or expensive instrumentation

needed • Adjustment to defined final protein concentra-

tion levels is possible • The fold concentration that is possible ranges

from low to high • Extensively used in the past

• Renaturation/refolding is necessary and may not always be possible

• Additional purification steps are required to remove precipitant and refolding agents

partial desiccation: A desiccant such as dry PEG or Sephadex beads is used to absorb water from a protein solution in a dialysis chamber

• Relatively easy to implement • No expensive or complex instrumentation

needed • Level of concentration can be controlled within

limits

• Typically, an overnight process • Water may not be the only protein solution

component absorbed by the desiccant • A small amount of polymer may transfer to

and contaminate the protein solution

Dialysis: A protein solution in a dialysis bag/chamber is equilibrated with a solution containing the protein buffer plus a high-MW polymer such as PEG

• Relatively easy to implement • No expensive or complex instrumentation

needed • Level of concentration can be controlled

within limits

• Typically, an overnight process • Water may not be the only protein solution

component lost in the process • A small amount of polymer may transfer to

and contaminate the protein solution

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on the identity of the detergent and that of the hosting and addi-tive mesophase lipid. The current study makes use of monoolein (9.9 MAG), monopalmitolein (9.7 MAG) and 9.9 MAG doped with 10% (wt/wt) cholesterol as mesophase lipids and DDM, lauryl maltose neopentyl glycol (LMNG), decyl maltose neopentyl glycol (DMNG) and LDAO as detergents. It was important therefore to understand the phase behavior of the assorted lipid–detergent combinations, as encountered and as used in this study. Qualitative indicators of phase identity in the case of the cubic mesophase include the fact that it is characteristically viscous, optically clear and non-birefringent1. Definitive characterization makes use of small-angle X-ray scattering (SAXS), which enables unambigu-ous phase identification and microstructure mensuration18,19. SAXS was used in the current detergent compatibility study in which samples were prepared at, or close to, full hydration and at increasing detergent concentration.

The results of the SAXS measurements are presented as isotherms in Figure 2 and in more detail in Supplementary Figures 1–5. Measurements with 9.9 MAG and DDM, the combination used to crystallize DgkA, showed expected behavior (Fig. 2a)16. The mesophase was of the cubic-Pn3m type with a lattice parameter (d100) of 100 Å in the absence of detergent. Small additions of DDM triggered the appearance of the cubic-Ia3d phase with a lattice parameter of ~150 Å. At 0.1 M DDM, the cubic phase was destabilized and the lamellar liquid crystal (Lα) emerged with a lattice parameter (d001) value of ~50 Å. The latter persisted in coexistence with the cubic-Ia3d phase up to a concentration of 0.2 M DDM. At no point in the investigated DDM concen-tration range was conversion entirely to the Lα phase observed.

The accepted view is that the lamellar phase alone is incompat-ible with 3D crystal growth by the in meso method and should be avoided15.

The other lipid–detergent combinations investigated included 9.7 MAG/DDM (PepTSt; Fig. 2b), 9.9 MAG-cholesterol/LMNG (β2AR; Fig. 2c), 9.9 MAG/LDAO (AlgE; Fig. 2d) and 9.9 MAG-cholesterol/DMNG (CFTR; Fig. 2e). These showed similar phase identity and sequence behavior to that described above for 9.9 MAG/DDM with some variation in the detergent concentra-tion range of phase stability.

The microstructure of the protein-laden mesophases used for crystallization and structure determination of the refer-ence proteins following rounds of reconstitution by the cubicon method was investigated. The SAXS data show that the protein and detergent loading that arises as a result of the cubicon treat-ment does not alter cubic phase behavior in a substantial manner (Supplementary Fig. 6).

The above measurements were carried out with the mesophase at or close to full hydration. The cubicon method involves pro-ducing fully hydrated mesophase in the presence of a considerable excess of aqueous buffer solution. To determine the identity and microstructure of the mesophases that form with an excess of the buffers used in the cubicon study, SAXS measurements were made with samples prepared using monoolein (9.9 MAG), mon-opalmitolein (9.7 MAG) and 9.9 MAG containing 10% (wt/wt) cholesterol. Without exception for all lipid/buffer combinations examined, only the cubic-Pn3m phase formed (Supplementary Table 1). However, the lattice parameter of that cubic phase was, in every case, larger than that observed in the earlier study

250

200

150

100

50

00.00 0.04 0.08

DDM (M)

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0.12 0.16 0.20 0.00 0.04 0.08

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0.12 0.16 0.20

Latti

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eter

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9.9 MAG + 10% (wt/wt) cholesterol

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a b

d e

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Figure 2 | Mesophase identity and microstructure of hydrated lipids as affected by detergent at 20 °C, determined using SAXS. (a–e) Dependence on detergent concentration of lattice parameters of the mesophases formed by mixtures of (a) DDM and 9.9 MAG at 40% (vol/vol) water; (b) DDM and 9.7 MAG at 50% (vol/vol) water; (c) LMNG and 9.9 MAG with 10% (wt/wt) cholesterol at 40% (vol/vol) water; (d) LDAO and 9.9 MAG at 40% (vol/vol) water; and (e) DMNG and 9.9 MAG with 10% (wt/wt) cholesterol at 40% (vol/vol) water. Concentration refers to the molar concentration (M) of detergent in the final mesophase. The vertical arrow in individual panels marks the detergent concentration in the mesophase following rounds of reconstitution as implemented by the cubicon method in the current study with DgkA (3.8 mM DDM) (a), PepTSt (1.8 mM DDM) (b), β2AR-T4L (1.5 mM LMNG) (c), AlgE (13.1 mM LDAO) (d) and CFTR (2.2 mM DMNG) (e). The identity of the different phases is as follows: ∆, cubic-Pn3m; ×, cubic-Ia3d; o, Lα. These measurements were made with water as the aqueous medium or lyotrope. The presence of buffers and salts at the concentrations encountered in routine biochemical and biophysical studies have little impact on mesophase identity and microstructure32. However, high (single-digit molar) salt concentrations can trigger phase transitions19.

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performed at or close to full hydration with water. Excess aque-ous phase that enables the mesophase to swell maximally and the presence of detergents that naturally lower interfacial curvature are likely contributors to the observed lattice expansion.

The SAXS data show that the cubic phases of the different lipids investigated were remarkably tolerant to added detergent. Thus, large amounts were accommodated without complete destabiliza-tion of the cubic phase. These quantities are considerably higher than those that could possibly come from the protein solutions used in cubicon trials. This means that the protein-enriched mes-ophase formed in the cubicon study described below should be capable of growing crystals by the in meso method.

Comparison with alternative methods. In addition to the cubi-con method described here, several alternative methods have been used to concentrate membrane proteins. A number of these meth-ods are described in Table 1, together with their advantages and disadvantages.

Limitations of the methodAs described previously, increased detergent concentrations will raise the complement of lamellar phase that, in turn, will change the physical properties of what amounts to a multiphase system. This can create difficulties in separating the protein-laden mes-ophase from the protein-depleted aqueous solution leading to loss of valuable protein. If the detergent load in the mesophase is too high, the cubic phase is destabilized completely and the resulting mesophase, which is usually of the lamellar type, is no longer useful for crystallization trials.

The cubicon method works well with the longer-chained lipids, 9.9 MAG and 9.7 MAG. These form a cubic phase that readily separates physically and in bulk from excess aqueous solution. With shorter-chain lipids, such as 7.7 MAG and 7.8 MAG that

are important host lipids for in meso crystallogenesis10,13,20–23, the mesophase that forms in equilibrium with excess aqueous solution is more fluid with a higher density compared with their longer-chained counterparts. This makes separating the protein-laden mesophase from the protein-depleted aqueous solution more challenging. A project is underway to address this issue and to deal with the equally challenging sponge variant of the cubic mesophase24.

The cubicon method involves multiple rounds of reconsti-tution. One might argue that it should be possible to ramp up mesophase protein concentration by using a larger volume of aqueous solution in a single reconstitution step. In practice, however, we have found that the efficiency of the partitioning process is considerably reduced when the aqueous solution-to-mesophase volume ratio is too high. In such circumstances, the protein tends to remain in the aqueous solution for longer mixing times and can be lost. Further, getting the mesophase to go from a fine dispersion, which arises when using an excess of aqueous solution, to a macroscopic phase-separated aggregate (Fig. 3) is technically more demanding and time-consuming.

Lipid Protein solution

MesophaseExcess aqueous

solutionProtein-laden cubic

mesophase

Mesophase +excess buffer dispersion

a b

c d

Figure 3 | Appearance of the lipid, aqueous solution and mesophase at various stages in the cubicon process. (a) Before commencing, mixing, the molten lipid and the protein solution in two separate syringes connected by a narrow-bore coupler appear as optically clear and colorless liquids. (b) The protein-laden mesophase in excess, protein-depleted aqueous solution appears as a white aggregate dispersed in a colorless liquid during the cubicon process. The inset shows an expanded view of the mesophase dispersion. (c) After each round of reconstitution, the protein-laden mesophase is physically separated from the bulk of the protein-depleted aqueous solution by way of the narrow-bore coupler. (d) The final step in the cubicon method involves converting the mesophase dispersed in a small amount of protein-depleted aqueous solution to the pure, optically clear and non-birefringent cubic phase. The entire cubicon process can be viewed directly in the online video (supplementary Video 1).

Lipid

+i ii iii iv v

Buffer Detergentmonomers

Protein−detergentmicelle

MesophaseProtein

Figure 4 | The cubicon method for raising membrane protein concentration in the bilayer of the cubic mesophase by sequential rounds of reconstitution. Step i: Lipid (dark red) and dilute, detergent-solubilized membrane protein solution are mixed, spontaneously forming the bicontinuous cubic mesophase with protein (and detergent) partitioning into the bilayer. Under the conditions used, excess protein-depleted aqueous solution (blue) exists in equilibrium with the fully hydrated mesophase (mesophase diagram). Step ii: Most of the excess protein-depleted aqueous solution that separates from the mesophase is removed. Step iii: Fresh, dilute protein solution is mixed with the mesophase in a step that facilitates partitioning of protein (and detergent) from the aqueous phase into the bilayer of the mesophase. Step iv: Most of the excess protein-depleted aqueous solution is removed. Steps iii and iv are repeated multiple times to effect rounds of reconstitution and a ramping up of protein concentration in the mesophase to a desired value. Step v: Excess protein-depleted aqueous phase is absorbed by mixing in a small amount of lipid to produce a homogeneous, optically clear, protein-enriched cubic phase suitable for crystallogenesis and other end uses.

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For this reason, we recommend using an aqueous solution-to-lipid/mesophase volume ratio of between 1.5 and 3.

Experimental designBefore embarking on crystallization trials, the membrane protein of interest must be shown to be stable and functional in solu-tion. Typically, this requires a series of screening studies in which pH and buffer, salt and detergent identity and concentration are optimized. Part of the optimization process involves identify-ing solution conditions in which the protein is soluble at high concentrations. In the field of membrane protein crystallization, 10 mg protein per ml is considered a reasonable concentration at which to begin crystallization trials. However, higher concentra-tions are always preferred for initial trials. If these do not provide structure-quality crystals, screening at lower concentrations is easily implemented.

When solutions at high protein concentrations are not forth-coming by standard methods, the cubicon method should be considered. For example, if 0.5 mg protein per ml is the highest concentration that can be achieved, the method can be used to incrementally ramp up protein concentration in the mesophase using the dilute starting solution. The detergent type and con-centration used to provide this dilute protein solution would have been identified on the basis of screening studies and will vary depending on the protein (see Table 2 for examples). The cubic mesophase is known to be compatible with a wide range of detergents16,17. Similarly, these detergents are compatible with the cubicon method, provided the concentration is not too high. Limiting concentration values are known for a number of deter-gents and related information on the detergent-carrying capacity of the cubic phase is included in the current Protocol (Fig. 2).

As noted previously, different hosting and additive lipids are used in in meso crystallization trials. These are an integral part of the crystallization screening process. For the membrane proteins

described in this Protocol, two different hosts (9.9 MAG, 9.7 MAG) and one additive lipid (cholesterol) were used. Specifically, 9.9 MAG was used with AlgE and DgkA, 9.7 MAG was used with PepTSt, and 9.9 MAG containing 10% (wt/wt) cholesterol was used with β2AR and CFTR. These specific protein–lipid combinations were arrived at on the basis of extensive screening trials described separately10–13. In the case of a protein that has not yet been crys-tallized, as applies in the case of CFTR in the current Protocol, the cubicon method must be implemented with a number of different hosts and additive lipids as part of a screening and optimization campaign.

The cubicon method involves an initial mixing of lipid with an excess of dilute protein solution to form the cubic mesophase. The protein preferentially partitions from solution into the bilayer of the mesophase. The highly viscous mesophase exists in equilibrium with excess protein-depleted aqueous solution, which is drawn off. Fresh dilute protein solution is then homoge-nized with the mesophase, whereupon the protein partitions into the mesophase. The excess protein-depleted aqueous solution is again drawn off. The last two steps are repeated several times until the protein concentration in the mesophase has risen to a suit-able level. The last cubicon step involves homogenizing the mes-ophase with a small amount of lipid to form the optically clear cubic mesophase, which can be used for crystallization or other end applications. A schematic overview of the method is pre-sented in Figure 4. A demonstration of the process can be viewed in an accompanying online video (Supplementary Video 1). All steps were carried out at room temperature (20 °C). unless otherwise noted. Although five proteins were examined in this study (Table 3; Anticipated Results), the protocol and details that follow refer primarily to CFTR. A detailed step-by-step protocol for the subsequent preparation of SAXS samples, setup of crystal-lization plates and harvesting of crystals is provided in Caffrey and Cherezov1.

MaterIalsREAGENTS

Monoolein (9.9 MAG) and monopalmitolein (9.7 MAG) (Nu-Chek Prep, cat. nos. M-239 and M-219) crItIcal To avoid degradation, lipids should be stored in the dark, under argon or nitrogen gas and at low temperature (−20 to −80 °C). Before use, lipids should be brought to and can be stored for short periods (up to 1 d) at room temperature.Lauryl maltose neopentyl glycol (LMNG; Anatrace, cat. no. NG310) ! cautIon Powdered LMNG is a respiratory sensitizer. It should be handled in a fume hood.

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Decyl maltose neopentyl glycol (DMNG; Anatrace, cat. no. NG322) ! cautIon Powdered DMNG is a respiratory sensitizer. It should be handled in a fume hood.n-Dodecyl-β-d-maltopyranoside (DDM; Anatrace, cat. no. D310A) ! cautIon Powdered DDM is a respiratory sensitizer. It should be handled in a fume hood.Lauryl dimethylamine-N-oxide (LDAO; Sigma-Aldrich, cat. no. 40263)Cholesterol (Sigma-Aldrich, cat. no. C8667)PEG 400 (Sigma-Aldrich, cat. no. 81172)

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table 2 | Composition of the buffers used in this study.

buffer protein lipid composition

A DgkA 9.9 MAG 10 mM Tris–HCl, pH 7.4, 100 mM NaCl, 1 mM TCEP, 0.02% (wt/vol) DDM

B PepTST 9.7 MAG 20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.009% (wt/vol) DDM

C β2AR 9.9 MAG + 10% (wt/wt) cholesterol 20 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% (wt/vol) LMNG, 10 µM Carazolol

D AlgE 9.9 MAG 50 mM Tris–HCl, pH 7.2, 150 mM NaCl, 0.03% (wt/vol) LDAO

E CFTR 9.9 MAG + 10% (wt/wt) cholesterol 40 mM Tris–HCl, pH 7.4, 150 mM NaCl, 100 mM arginine, 5 mM Mg-ATP, 3 mM TCEP, 10% (vol/vol) glycerol, 0.01% (wt/vol) DMNG

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Sodium sulfate (Na2SO4; Sigma-Aldrich, cat. no. 239313)Sodium hydroxide (NaOH; Sigma-Aldrich, cat. no. S8045) ! cautIon NaOH is corrosive; wear appropriate personal protective equipment.Acetic acid (Sigma-Aldrich, cat. no. 320099) ! cautIon Acetic acid is flammable and corrosive; work in a fume hood and wear appropriate personal protective equipment.EDTA (Sigma-Aldrich, cat. no. ED2SS) ! cautIon EDTA can be harmful; wear appropriate personal protective equipment.Trizma base (Tris) (Sigma-Aldrich, cat. no. T1503)1,4-Butanediol (Sigma-Aldrich, cat. no. 493732)Lithium chloride (LiCl; Sigma-Aldrich, cat. no. L9650)Sodium citrate (Sigma-Aldrich, cat. no. S4641)2-Methyl-2,4-pentanediol (MPD; Sigma-Aldrich, cat. no. 68340)HEPES (Sigma-Aldrich, cat. no. H4034)Sodium acetate (Sigma-Aldrich, cat. no. S2889)Hydrochloric acid (HCl; Sigma-Aldrich, cat. no. 07102) ! cautIon HCl is corrosive and harmful; work in a fume hood and wear appropriate personal protective equipment.Ammonium phosphate monobasic (NH4H2PO4; Sigma-Aldrich, cat. no. 216003)Imidazole (Sigma-Aldrich, cat. no. I2399) ! cautIon Imidazole can be harmful, corrosive and a health hazard; work in a fume hood while preparing buffers, and wear appropriate personal protective equipment.RunBlue Rapid SDS run buffer, 20× (Expedeon, cat. no. NXB14500)Tris(2-carboxyethyl)phosphine HCl (TCEP; Sigma-Aldrich, cat. no. C4706)Carazolol (Sigma-Aldrich, cat. no. 53787)Arginine (Sigma-Aldrich, cat. no. A5006)Adenosine 5´-triphosphate magnesium salt (Mg-ATP; Sigma-Aldrich, cat. no. A9187)Glycerol (Sigma-Aldrich, cat. no. G5516)InstantBlue Coomassie stain (Expedeon, cat. no. ISB1L)Tris-glycine sample buffer (2×) (Novex, cat. no. LC2676)PageRuler broad-range unstained protein ladder (Thermo Fisher, cat. no. 26630)Bis–tris propane (Melford, cat. no. B7510)

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Sodium chloride (NaCl; Fisher Scientific, cat. no. BP358-212)Methanol, technical grade (Hazardous Materials Facility, Trinity College Dublin; equivalent-grade methanol is available from VWR, cat. no. 80700-774) ! cautIon Methanol causes acute toxicity and is a health hazard. It is flamma-ble; work in a fume hood and wear appropriate personal protective equipment.Chloroform, HPLC grade (Hazardous Materials Facility, Trinity College Dublin; equivalent-grade chloroform is available from VWR, cat. no. BDH83626.400) ! cautIon Chloroform causes acute toxicity and is a health hazard; work in a fume hood and wear appropriate personal protective equipment.Liquid nitrogen (Hazardous Materials Facility, Trinity College Dublin; equivalent-grade liquid nitrogen is available from BOC or other suppliers) ! cautIon Liquid nitrogen is a refrigerated liquefied gas. It can cause cold burns; wear appropriate personal protective equipment.

EQUIPMENTTools for preparing SAXS samples

Borosilicate glass capillaries (length, 80 mm; outer diameter, 1 mm; 0.01-mm thick; Hampton Research, cat. no. HR6-122)Turbo Set 90 welding torch (Wigam, cat. no. 10001013)Araldite 5-min epoxy (Everberg; was bought in a local hardware store)

Tools for preparing the lipid cubic phase, setting up crystallization plates and harvesting of crystals

Analytical balance with submilligram sensitivity (Sartorius, model no. CP64)Wash bottle with fine tip filled with methanol, used to clean syringes, needles, couplers and ferrulesGloves to protect hands while working with acids and organic solvents (Fisher Scientific, cat. no. 19-130-1597)1.5-ml Eppendorf tubes and racksBenchtop centrifuge (Spectrafuge 24D; Labnet International, cat. no. C2400)Assortment of Eppendorf pipettes and tipsCompressed nitrogen gas or air for drying syringes, needles, ferrules and couplers (Hazardous Materials Facility, Trinity College Dublin; equivalent-grade nitrogen gas is available from BOC, cat. no. 44-Cert)Standard glass plates (127.8 × 85.5 mm2, 1 mm thick) and no. 1.5 glass covers (112 × 77 mm2, 0.15 mm thick) (Marienfeld, cat. nos. 1527127092 and 01029990911)

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table 3 | Summary of the conditions used and the results obtained upon implementing the cubicon method with five integral membrane proteins.

experimental details Dgka peptst b2ar alge cFtr

Protein conc. in stock solution (mg/ml) 12 10 45 20 0.5a

Protein conc. in dilute ‘starting’ solution (mg/ml) 0.5 0.5 1 1 0.5a

Protein conc. in ‘equivalent’ stock solution (mg/ml) 12 10 45 20 18

Protein conc. in ‘final’ mesophase (mg/ml) 4.8 5 18 8 7.2

No. of rounds of reconstitution 12 12 12 10 17

Protein in excess aqueous solution after rounds of reconstitutionb

ND ND ND Trace ND

Fold concentrationc 24 20 45 20 36

Bufferd A B C D E

Detergent DDM DDM LMNG LDAO DMNG

Lipid 9.9 MAG 9.7 MAG 9.9 MAG + 10% (wt/wt) cholesterol

9.9 MAG

9.9 MAG + 10% (wt/wt) cholesterol

Resolution of cubicon structurese (Å) 3.1 (2.7–3.3) 2.4 (–) 3.2 (2.4–3.8) 2.7 (–) NANA, not applicable.aThe concentration of CFTR was estimated by comparison with a Coomassie-stained BSA loading series on an SDS-PAGE (supplementary Fig. 7). bND, none detected. Trace, estimated at 0.15% of total protein based on a comparison with a Coomassie-stained AlgE loading series on an SDS-PAGE (supplementary Fig. 11). cFold concentration was calculated as (protein conc. in equivalent solution used to make final mesophase)/(protein conc. in dilute starting solution). See worked example in Step 26. dSee table 2 for buffer composition. eNumbers in parentheses correspond to the resolution or resolution range of refer-ence protein structures reported in the PDB that were obtained by conventional reconstitution of a similar construct into the same lipid.

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Rain-X rain repellent (Shell Car Care, cat. no. 80199200).Perforated double-stick spacer tape (112 × 77 mm2, 140 µm thick, with perforations 6 mm in diameter) (Saunders, cat. nos. 9500PC and 9009)Gryphon LCP liquid-handling system (Art Robbins Instruments, cat. no. 620-3000-20)Heat block at 42 °C to melt the lipidOptional wheat pack for preheating syringes to 42 °CTwo 100-µl Hamilton gas-tight syringes with removable needles and Teflon ferrules (Hamilton, cat. no. 81030); syringe coupler fabricated as described in Cheng25. Syringe couplers are available as part of LCP kits from Rigaku (cat. no. SKU:1006904), MiTeGen (cat. no. M-R1006905) and Art Robbins Instruments (cat. no. 620-1200-03)RunBlue SDS precast polyacrylamide gels, 12% (wt/vol), 12 well, 10 × 10 cm (Expedeon, cat. no. NXG01212)RunBlue electrophoresis gel tank (Expedeon, cat. no. NXE00014)PowerPac Basic (Bio-Rad, cat. no. 1645050)Hand-held roller or brayerMicroscope with polarizer and rotating analyzerGlass cutters (TCT, cat. no. 633657)Fine-point tweezers (Ted Pella, cat. no. 5667)Magnetic wand (Hampton Research, cat. no. HR4-729)

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Harvesting cryoloops (MiTeGen, cat. nos. M2-L18SP-20, M2-L18SP-30 and M2-L18SP-50)Covered Dewar filled with liquid nitrogen (Hampton Research, cat. no. HR4-662)Storage and shipping Dewar (Taylor Wharton Shippers; Marathon Products)Pucks to house mounted crystals (can be obtained from Crystal Positioning Systems or MiTeGen)

REAGENT SETUPA step-by-step video demonstration of the cubicon method, as detailed in this Protocol, is provided in Supplementary Video 1. Details regarding the setup of equipment are provided in Caffrey and Cherezov1.Buffers Buffers should be made up as described in Table 2.Proteins to be concentrated In this protocol, we use purified CFTR, β2AR, PepTSt, DgkA and AlgE. Details regarding the production, purification and crystallization of the proteins used in this study are provided in the Supplementary Methods. Materials to start the rounds of reconstitution include protein solution (CFTR, 0.5 mg/ml; β2AR, 1 mg/ml; PepTSt, 0.5 mg/ml; DgkA, 0.5 mg/ml; AlgE, 1 mg/ml) held on ice and lipid (9.9 MAG, 9.7 MAG and 9.9 MAG with 10% (wt/wt) cholesterol).Lipids The preparation of the 9.9 MAG with cholesterol mixture is described in Caffrey and Cherezov1. Lipids can be stored at room temperature until time of use.

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proceDurepreparation of tools and equipment ● tIMInG 30 min1| Clean tools and equipment as described in the Equipment Setup and supplementary Video 1. crItIcal step Clean the syringes, plungers, couplers and ferrules in a fume hood with methanol from a wash bottle with a fine tip. Using a flow of compressed nitrogen gas or air, thoroughly dry all coupled-syringe mixer parts.

loading of the coupled-syringe mixing device with lipid and dilute protein solution ● tIMInG 15 min2| Place lipid and pipette tips on a heat block at 42 °C. crItIcal step Warm the lipid only until it is fully melted. Extended heating can cause the lipid to degrade. We typically work with 50-mg quantities of lipid at a time.

3| Using a pipette, transfer the protein of interest (in this example we use 32 µl of CFTR solution at 0.5 mg/ml) to one of the Hamilton syringes, loading from the front end of the syringe while slowly withdrawing the plunger. crItIcal step It is important to avoid trapping air bubbles in the protein solution at this step. The protein solution contains detergent that can stabilize bubbles. Remove air bubbles trapped in the solution by rapid up-and-down movements of the plunger in the barrel while holding the syringe vertically (demonstrated in supplementary Video 1).? troublesHootInG

4| Record the volume of the protein solution in the syringe. It should be ~32 µl. Move the protein solution until it is level with the top of the syringe barrel. The position of the protein solution can be tracked by looking through the window at the syringe termination or by looking down the open end of the barrel to view the meniscus of the protein solution in the syringe. When the protein solution is level with the top of the syringe barrel, the meniscus is flat and, in the right light, characteristically reflective.

5| Using a pipette and a prewarmed pipette tip, transfer 15 µl of molten lipid to the second Hamilton syringe. Remove air bubbles trapped in the molten lipid as described in Step 3. Record the volume of lipid (15 µl). Move the lipid toward the top of the glass barrel as in Step 4. crItIcal step We recommend using a 1:1.5–3.0 volume ratio of lipid and protein solution during the rounds of reconstitution. crItIcal step Work quickly to avoid solidification of the lipid, especially if the ambient temperature is <20 °C. Upon solidification, the optically clear molten lipid will turn white.? troublesHootInG

6| Attach the ferrules to the prewarmed coupler. Attach the coupler to the lipid syringe and tighten. crItIcal step Finger-tighten the syringe and the coupler to avoid leaking. Leakage will happen if the device is under-tightened. However, over-tightening can deform the ferrule and/or break the glue seal between the glass syringe and the steel barrel termination. In either case, the device will leak upon mixing. It is recommended to perform several practice

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runs with lipid and protein-free buffer to gain familiarity with the device, the materials and the method—before using precious test protein sample.? troublesHootInG

7| Slowly force the molten lipid into the coupler until the molten lipid emerges at the coupler’s end. Reverse the process until the lipid becomes level with the end of the coupler needle. crItIcal step To prevent the lipid from solidifying upon transfer, make sure that the temperature of the syringe and the coupler is at least 20 °C.

8| Connect the protein-containing syringe to the other side of the coupler and finger-tighten the device (Fig. 3a). crItIcal step Be careful to only finger-tighten the device. Under- or over-tightening may cause it to leak.

performing of the first stage of the cubicon process ● tIMInG 10 min 9| Push the plunger in the lipid syringe to move the molten lipid into the protein syringe. Push the plunger in the protein syringe to pass material into the lipid syringe.

10| Repeat the mixing process for 2–5 min at a rate of ~140 strokes (70 strokes forward and 70 strokes backward) per min. Be sure to move the plunger to the bottom of the barrel during each mixing cycle to ensure homogeneous mixing of the contents of the two syringes. The net effect of the mixing process is to create the cubic mesophase and to bring about partitioning of the protein from the aqueous solution into the bilayer of the mesophase. Because an excess of aqueous solution is used, a two-phase system forms upon mixing. One phase is the protein-laden mesophase, which appears as a white aggregate. The other is the protein-depleted aqueous solution. The two exist in equilibrium as a dispersion of mesophase in an aqueous solution (Fig. 3b). They can be separated physically. crItIcal step A mixing rate of ~140 strokes per min is recommended. A lower rate can result in a reduced partitioning efficiency and a substantially higher rate can raise the sample temperature because of frictional heating25. A mixing duration of 2–5 min per cubicon round is recommended. In our experience, partitioning is complete within a couple of minutes at 140 strokes per min. However, when starting with a new protein target, mixing time should be investigated and optimized.? troublesHootInG

11| Transfer the dispersion to one of the syringes in the coupled-syringe mixing device and record its total volume. It should be close to the sum of the molten lipid and protein solution volumes minus 2 µl, which is the dead volume of the coupler. In our experiment, the measured volume was 47 µl.

separation of the mesophase from the excess aqueous solution ● tIMInG 5 min12| Separate the white mesophase aggregates from the excess aqueous solution by using the narrow-bore coupler effectively as a filtering device. The separation process benefits from the natural stickiness of the mesophase, which tends to adhere to itself and to separate naturally from the aqueous solution. By moving material back and forth slowly between the two syringes, eventually most, if not all, of the white mesophase can be collected in one syringe, with the aqueous solution in the other (Fig. 3c, supplementary Video 1). crItIcal step Cleanly separating the cubic mesophase from the aqueous solution is an essential step in this Protocol. It requires practice, which can be gained inexpensively by using molten lipid and protein-free buffer solution in advance of working with precious test protein. crItIcal step Every effort should be made to separate as much of the protein-depleted aqueous solution as possible from the protein-laden mesophase. The knowledge that the 9.9 MAG mesophase, when fully hydrated at 20 °C, is ~2/5 aqueous solution and ~3/5 lipid by volume2,15 allows one to calculate the volume of mesophase expected. In the current example, 15 µl of lipid was used initially and thus (15 × 5/3 =) 25 µl of fully hydrated mesophase is expected. If all the excess aqueous solution were separated successfully, the mesophase volume in the mesophase syringe would read somewhere between 25 and 30 µl. Typically, we find that recorded mesophase volumes at this stage in the cubicon process are 5–10% greater than the expected values. crItIcal step We noticed that the detergent LDAO, when used at concentrations well above its critical micelle concentration (CMC; 1–2 mM, 0.23 g/l)25, caused the mesophase to form a dispersion of fine particles. This type of dispersion requires more time and effort to effect a clean separation of phases. When new lipids, detergents and/or high detergent concentrations are used, phase behavior should be monitored carefully and the appropriate exploratory work done in advance with protein-free solutions.? troublesHootInG

13| Uncouple the syringes, leaving the coupler attached to the mesophase-containing syringe. Using a pipette, remove the protein-depleted aqueous solution from the other syringe. This completes the first round of the cubicon procedure. crItIcal step Save the protein-depleted aqueous phase at −20 or −80 °C for protein analysis to monitor the progress of the partitioning process. Protein quantification can be performed by SDS-PAGE analysis, as described below.

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repetition of the cubicon round ● tIMInG 15 min per round14| Initiate the second stage in the cubicon procedure. This involves mixing the separated protein-laden mesophase produced in Step 12 with fresh dilute protein solution. Specifically, load 32 µl of protein solution into a clean, dry 100-µl Hamilton syringe, as described in Step 3. Adjust the protein solution in the syringe to the top of the barrel. Combine the two syringes and finger-tighten, as in Step 8. Mix the contents of the two syringes to effect partitioning of protein out of the aqueous solution and into the mesophase bilayer, as in Steps 9 and 10. Separate the protein-laden mesophase from the protein-depleted aqueous solution, as in Step 12. Uncouple the syringes and remove and save the aqueous solution, as in Step 13. This completes the second round of the cubicon procedure.? troublesHootInG

15| Repeat Step 14 until the desired concentration of protein in the mesophase is reached. In the case of CFTR, anywhere from 10 to 26 rounds of reconstitution have been used. crItIcal step Save the protein-depleted aqueous solution after each round for protein analysis to monitor progress of the partitioning process, as described in Step 13. crItIcal step Throughout the procedure be careful to note and to act immediately on any leakage of the coupled syringes.? troublesHootInG

production of an optically clear cubic mesophase ● tIMInG 15 min16| After completing the last round in the cubicon procedure (Step 15), record the volume of the protein-laden hydrated mesophase, having separated as much of it as possible from the protein-depleted aqueous solution. In our experiment, the volume was 30 µl. crItIcal step At the final round of the cubicon procedure, try to separate and remove as much as possible of the aqueous solution from the mesophase dispersion. By doing so, less molten lipid is needed to convert the mesophase dispersion to the optically clear cubic phase. This ensures that the final concentration of protein in the mesophase is maximized.

17| Calculate the volume of additional molten lipid that must be combined with the protein-laden mesophase dispersion (30 µl, in our experiment) so as to produce an optically clear cubic mesophase at or close to full hydration. Assuming that the entire 15 µl of the starting lipid (Step 5) is in the final mesophase dispersion, the latter contains (30–15 =) 15 µl of aqueous solution. To convert this 15 µl of aqueous solution entirely to a fully hydrated cubic phase (with 2/5 being aqueous solution and 3/5 being lipid) a total of (15 × 3/2 =) 22.5 µl of lipid is needed. With 15 µl of starting lipid already in the dispersion, an additional (22.5–15 =) 7.5 µl of lipid must be added to the mesophase dispersion. crItIcal step Unlike 9.9 MAG, which hydrates fully at 40% (wt/wt) water and 20 °C, 9.7 MAG achieves full hydration at 50% (wt/wt) water26. Accordingly, the ratio of aqueous solution and lipid required to produce a fully hydrated and optically clear cubic phase prepared with 9.7 MAG is 1:1.

18| Add the calculated volume of molten 9.9 MAG containing 10% (wt/wt) cholesterol to a clean, dry and prewarmed syringe, as in Step 5. Couple the syringe to the mesophase-containing syringe and mix their contents to restore the protein-laden mesophase dispersion to the pure cubic phase state, which is optically clear and characteristically viscous. The markings on the barrel can be seen through the optically clear mesophase if it has been properly formed (Fig. 3d).? troublesHootInG

19| Check that the mesophase is non-birefringent by viewing it in the syringe barrel or on a glass slide between crossed polarizers. Non-birefringence is a characteristic of the cubic phase1. The protein-laden mesophase is now ready for use in crystallization trials1 or other applications.

analysis of the protein-depleted aqueous phase ● tIMInG 3–6 h20| Assemble the RunBlue electrophoresis gel tank and fill it with 1 × SDS running buffer.

21| Mix 5 µl of the protein-depleted aqueous solution that was saved after each cubicon round with 5 µl of 2× Tris-glycine SDS sample buffer at 20 °C and load on a 12% (wt/vol) SDS precast polyacrylamide gel. PageRuler broad range unstained protein ladder standards and samples containing 0.2, 0.5 and 1 µg of reference protein should be included on the gel.

22| Connect the gel tank to a power pac and run the gel for 40 min at 150 V at 20 °C.

23| Stain the gel with InstantBlue Coomassie stain for 1 h.

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24| Destain the gel with milli-Q water for 2–5 h. Replace the water with fresh water once or twice for more effective destaining.

25| Quantify the amount of protein in the protein-depleted aqueous solutions by comparing the stain level for protein samples in the gel with that for reference protein in the loading series. In our experiment, no protein was observed in the aqueous solutions following cubicon rounds with CFTR, consistent with complete partitioning of the protein into the mesophase by the cubicon method. Examples of SDS-PAGE data for the reference membrane proteins described in the Anticipated Results are provided in supplementary Figures 7–11.? troublesHootInG

calculation of fold concentration ● tIMInG 5 min26| Calculate the fold concentration as follows: In our experiment, the stock CFTR solution had a protein concentration of 0.5 mg/ml. A total of 540 µl of this starting solution was used to perform 17 rounds of reconstitution with 15 µl of 9.9 MAG containing 10% (wt/wt) cholesterol, hereafter referred to as lipid. At the end of the procedure, 7.5 µl of lipid was added to convert the mix to the pure cubic phase. The total volume of final mesophase was carefully measured at 37.5 µl. Assuming that all the protein (540 µl at 0.5 mg/ml = 0.27 mg CFTR) was reconstituted uniformly in the 37.5-µl mesophase by the cubicon method, the concentration of protein in the final mesophase was 0.27 mg/0.0375 ml = 7.2 mg/ml. We next calculate the protein concentration in the so-called ‘equivalent’ stock solution. This refers to a protein solution that will make pure mesophase (in other words a single-phase system with no excess aqueous solution) at 7.2 mg protein per ml in the final mesophase in just a single round of reconstitution. Pure mesophase made with lipid is prepared at a protein (or buffer) solution/lipid volume ratio of 2:3 (or 4:6). The calculations that follow are simplified by working with a mesophase volume of 1 ml. Therefore, 1 ml of the mesophase was generated by homogenizing 0.4 ml of protein solution with 0.6 ml of lipid. If this 1 ml of final mesophase has a protein concentration of 7.2 mg/ml, it contains 7.2 mg of protein. This 7.2 mg of protein originated from the 0.4-ml stock solution used to make the final mesophase. Thus, the concentration of protein in what we refer to as the ‘equivalent’ stock solution used to make the pure mesophase in a single round of reconstitution was 7.2 mg/0.4 ml = 18 mg/ml. The starting stock solution pre-cubicon was 0.5 mg protein per ml. It produced a final mesophase at 7.2 mg protein/ml post cubicon. Therefore, the fold concentration, defined as the ratio of protein concentration in the ‘equivalent’ and starting dilute solutions, is 18 mg/ml/0.5 mg/ml = 36. Thus, the cubicon method enabled the creation of a mesophase at 7.2 mg protein per ml with a starting solution at a concentration of 0.5 mg/ml. An equivalent mesophase could be generated by the standard, single-round reconstitution method but only with a starting solution some 36-fold higher in protein concentration at 18 mg/ml. This highlights the power of the cubicon method. Values used in calculating fold concentration for the other proteins in this Protocol are included in supplementary table 2.

? troublesHootInGTroubleshooting advice can be found in table 4.

table 4 | Troubleshooting table.

steps problem possible reason solution

Step 3 Bubbles cannot be removed after loading the Hamilton syringe with protein solution

The detergent stabilizes the bubbles

If bubbles form and cannot be removed by rapid up-and-down movements of the plunger in the syringe barrel, it is best to transfer the protein solution back to the Eppendorf tube, to centrifuge it briefly to break the foam and to begin again

Steps 5 and 6

The lipid solidifies upon transfer to the Hamilton syringe

The temperature of the syringe is too low

As noted, the molten lipid from the heat block at 42 °C should be transferred to a syringe that is at a temperature of at least 20 °C. If ambient or room temperature is 20 °C or slightly above, the syringe is warm enough and problems with lipid freezing should not arise. If, however, ambient temperature is <20 °C, special care should be taken to ensure that the syringe and the coupler are prewarmed to, and are maintained at, 20 °C (or slightly above) from the time molten lipid is transferred to the syringe until the mesophase is formed. Once the mesophase has formed, ambient temperature—within limits—is no longer a concern. The syringe and coupler can be prewarmed by placing them on the heat block at 42 °C for 10–20 min or in a microwave-warmed wheat bag ahead of loading with molten lipid.

(continued)

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table 4 | Troubleshooting table (continued).


Steps 5 and 6

The lipid solidifies upon transfer to the Hamilton syringe

The temperature of the syringe is too low

Note, however, that preheating the steel terminator for extended periods is not recommended; the glue seal can come undone. If ambient temperature is particularly low (16–18 °C), it is very important to work quickly to minimize the time between loading the syringe and making the mesophase. If the lipid does solidify, the coupler will plug and the solid lipid will be difficult (impossible) to move in the syringe barrel. In this case, the lipid can be melted by warming it with a tissue moistened with hot water or with the warmed wheat pack. Should neither of these methods work, the syringe is best disassembled and carefully washed with methanol and dried. If excess force is applied to the plungers in an attempt to use the solidified lipid for mesophase formation, the coupler will probably leak and the glue seal on the syringe termination will probably break. This will require replacing the syringes and possibly the Teflon ferrules

Step 10 Leakage at the coupler Loose connection, damaged ferrules

Finger-tighten the connection between the coupler and the syringes and try again. If this fails and leakage continues, replace the ferrules with new ones. The couplers themselves are made of steel and, in our experience, never fail. Although it is important to tighten the coupler connections to the syringes adequately, it is just as important not to over-tighten them. If this happens, the Teflon ferrules can deform excessively and leak

Leakage at the syringe barrel termination

Broken seal. The steel termination and the glass barrel of the syringe are held together by glue. The seal can break through mechanical failure because of rough handling and over-tightening. It can also fail with age, and exposure to harsh solvents and high temperatures

If the seal breaks, the syringe should be disassembled and replaced with a new one. It is difficult to anticipate when a syringe will leak, so it is important to monitor the condition of the syringes and to have replacements in stock

Leakage at the plunger Plunger failure The plunger in gas-tight syringes is capped with a Teflon tip. Over time, the Teflon tip can deform to such an extent that it leaks. When this happens, aqueous solution and/or mesophase will be seen behind the Teflon tip around the steel plunger. Replacing the plunger with a new one usually solves the problem. In the unusual event that the glass barrel becomes worn, a new syringe is required

Step 12 Difficulty in separating the mesophase from the aqueous solution

The mesophase exists as a fine dispersion

This problematic behavior was pronounced with the detergent LDAO. Instead of forming clumps or aggregates of mesophase that naturally stick together, a fine dispersion was produced. Using LDAO, it took considerable time and effort to get the mesophase to form a macroscopic aggregate and to separate from the protein-depleted aqueous solution. Rapidly moving (snapping) the plunger in the syringe containing residual, finely dispersed mesophase toward the coupler end of the syringe generally had the effect of transferring the mesophase to the other syringe. Subsequent gentle movement of the plunger in the receiving syringe facilitated collection of the finely dispersed mesophase together in the receiving syringe with the protein-depleted aqueous solution in the other (supplementary Video 1).

(continued)

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Step 12 Difficulty in separating the mesophase from the aqueous solution

The mesophase exists as a fine dispersion

Where problems associated with a finely dispersed mesophase are encoun-tered, it is advisable to work at the lowest possible detergent concentration at which the protein remains stable and in solution. Alternatively, other detergents should be investigated. As noted, effective mesophase separation is key to the cubicon method. Mastery of the method comes with practice and can be acquired conveniently by following the procedures demonstrated in supplementary Video 1 while working with protein-free buffer. Ideally, the buffer used should have the same composition as the buffer in which the test protein is dissolved

The mesophase and the aqueous phase become inseparable

Formation of the lamellar phase, caused by detergent overload

The cubic phase can accommodate finite amounts of detergent before it destabilizes, usually forming the lamellar liquid crystal or Lα phase (Development of the protocol section, Fig. 2). The amount tolerated depends on, among other things, the identity of the detergent and of the mesophase-forming lipid. In our experience, most detergents that are commonly used in the field are compatible with the cubic phase and are likely to be compat-ible with routine application of the cubicon method. However, if too much detergent is carried over with the protein and the lamellar phase forms, it will be immediately obvious. The lamellar phase exists as a relatively stable liquid dispersion of multilamellar vesicles that is not at all viscous. The vesi-cles have weak adherent properties and only with time do they settle and phase-separate. Further, when examined with a microscope between crossed polarizers, the lamellar phase is birefringent. By contrast, the cubic phase is optically isotropic and non-birefringent. Problems associated with lamellar phase formation can usually be avoided by reducing the detergent concentra-tion in the protein solution to values close to the detergent’s CMC

Step 15 Syringe failure Excessive use, misuse

The cubicon method requires heavy use of Hamilton syringes. For example, a single cubicon experiment can involve as many as (140 strokes per min × 5 min × 20 rounds =) 14,000 back-and-forth passages of the plunger in each syringe. Syringe barrels are made of glass and the gas-tight seal is provided by contact between the Teflon tip on the plunger and the glass wall, which is expected to be of low friction. As noted, syringes can leak as a result of plunger tip deformation brought about by excessive use. The problem is easily dealt with by replacing old with new plungers on a regular basis. On one occasion, during the course of a cubicon experiment, the mesophase, which is normally white in appearance, became grayish black. We attributed this to improper use of the syringe mixer, in which the plungers were activated at an angle with respect to the long axis of the syringe barrels. Thus, in addition to Teflon-on-glass contact, it is probable that the steel plunger scraped along the glass barrel, generating microscopic metal particles through abrasion. In this instance, an old syringe may have been used, exacerbating the problem. Although this was a one-off event, it is recounted here to remind experimenters of the need to deploy equipment that is in good working order and to use it properly

Step 18 Cloudy mesophase Excess protein solution, excess lipid, excess deter-gent in protein solution, trapped air, insufficient mixing and tem-perature issues

The problem of a cloudy mesophase and how to solve it has been dealt with in Caffrey and Cherezov1. However, of the many possible reasons for cloudiness, temperature was not discussed in Caffrey and Cherezov1. Because it is an issue that has been encountered by many users of the in meso method, it is important and will be expanded on here. The protocol, as described, assumes that ambient temperature is regulated at 20 °C. When ambient temperature is too high, the mesophase, which was prepared at a composition at or just below full hydration in a pure, optically clear state, is no longer stable as a single phase. Rather, the mesophase sheds water or aqueous solution, as dictated by the corresponding temperature–composition phase diagram7.

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Step 18 Cloudy mesophase Excess protein solution, excess lipid, excess detergent in protein solution, trapped air, insufficient mixing and temperature issues

When the resulting two-phase system, consisting of fully hydrated mesophase and excess aqueous solution, is homogenized in the syringe mixer, a dispersion is created, which scatters light and is cloudy in appearance. The rise in ambient temperature needs to be only a few degrees Celsius for the effect to be seen. It is possible to reverse the effect simply by touching the coupler of the syringe mixing device to a cold object, such as a piece of ice, for a few seconds while continuing to mix the mesophase. The drop in mesophase temperature by a few degrees is usually enough to cause the excess aqueous solution to be reabsorbed by the mesophase and for the system to return to its original, intended optically clear pure cubic phase. Continuing the mixing process in a walk-in cold room at 4 °C or thereabouts or in the stream of cool air from an air conditioner works just as well as using ice. Be careful, however, not to over-cool the system, in which case the solid, lamellar crystalline (Lc) phase may form. The cloudiness in the mesophase can return when the sample rewarms. However, usually this is a slow-enough process that there is time to use the optically clear material to set up crys-tallization trials. Restoring the optical clarity of the mesophase can also be done without changing the sample temperature. To do so, additional molten lipid must be added to, and homogenized with, the cloudy mesophase, as described above for Steps 16–19. It is important to note that the presence of cloudiness because of a small amount of excess aqueous solution should not have an impact on the outcome of subsequent crystallization trials. After all, screening is done in the presence of a vast excess of precipitant solution; typically, 50 nl of mesophase and 800 nl of precipitant solution are used. However, excessive cloudiness leading to bulk-phase separation should be avoided. When such a multiphase dispersion is used to set up crystallization trials, some wells (screen conditions) will be loaded correctly with protein-laden mesophase, whereas others will receive protein-depleted aqueous solu-tion, compromising the trial. If ambient temperature control is not available in the laboratory where the cubicon method is being used, the possibility of the sample being prepared at temperatures below the desired 20 °C also exists. This issue, as applied to molten lipid solidifying ahead of mesophase preparation, has been addressed in this table in the entry for Steps 5 and 6. If, however, it has been possible to create the mesophase in its expected, optically clear form at 20 °C, and then lowering the temperature to <20 °C is usually not a problem with the default lipid, monoolein (9.9 MAG). This is because the cubic mesophase undercools naturally and remains in a long-lived metastable state, certainly at temperatures as low as 4 °C in the cubic phase, at which temperature crystallization trials can be conducted reliably3,23. The 7.9 and 9.7 MAGs are lipids that form a stable cubic phase that can be used in crystallization trials at reduced temperatures26,33

Steps 10, 12, 14, 15 and 25

Residual protein in the separated aqueous solution

Incomplete homog-enization of pro-tein solution and molten lipid or mesophase

The protocol calls for the mixing of molten lipid or mesophase with protein solution in the syringe mixing device at a rate of 140 complete strokes per min for 2–5 min to ensure effective homogenization and partitioning of mem-brane protein into the bilayer of the mesophase. Reducing the rate and/or duration of mixing significantly from the recommended values runs the risk of compromising the homogenization and partitioning processes, leading to protein remaining in the phase-separated aqueous solution. Not using the full range of plunger motion in the syringe during the mixing cycle similarly will lead to incomplete homogenization and transfer of protein to the mesophase bilayer. In this regard, it is important for the experimenter to feel in his/her fingertips the plunger strike or contact with the steel syringe termination at the end of each mixing stroke. This ensures that the entire contents of the syringe have been forced through the narrow-bore coupler and into the receiving syringe for thorough homogenization

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● tIMInGStep 1, preparation of materials, tools and equipment: 30 minSteps 2–8, loading of the coupled-syringe mixing device with lipid and dilute protein solution: 15 minSteps 9–11, performing of the first step of the cubicon process: 10 minSteps 12 and 13, separation of the mesophase from the excess aqueous solution: 5 minSteps 14 and 15, repetition of the cubicon round: 15 min per roundSteps 16–19, production of an optically clear cubic mesophase: 15 minSteps 20–25, analysis of the protein-depleted aqueous phase: 3–6 hStep 26, calculation of fold concentration: 5 min

antIcIpateD resultscystic fibrosis transmembrane conductance regulatorA high-resolution crystal structure for CFTR is needed to understand its mechanism of action and for rational drug design and discovery. To this end, the in meso method is being investigated as a means for growing crystals of this medically important channel. Because of instability, the protein aggregates at concentrations above ~0.5 mg/ml9. To raise the protein concentration to a target value of 8 mg/ml in the mesophase that enters crystallization trials, corresponding to 20 mg/ml in the starting protein solution, the cubicon method, as demonstrated successfully above, was implemented. The protein, dissolved in Buffer E (table 2), was reconstituted into 9.9 MAG doped with 10% (wt/wt) cholesterol. Seventeen rounds of reconstitution with a total of 22.5 µl of lipid and 540 µl of protein solution at ~0.5 mg/ml effected an estimated 36-fold protein concentration, producing a mesophase with 7.2 mg of CFTR per ml and 2.2 mM DMNG. This corresponds to a starting protein solution of 18 mg CFTR per ml. The absence of protein in the excess aqueous solution removed following reconstitution rounds corroborates this estimate (supplementary Fig. 7). The final



Steps 10, 12, 14, 15 and 25

Incomplete separation of the aqueous solution and the mesophase

Given the amphiphilic nature of a membrane protein and the vast bilayer reservoir available to it for reconstitution in the mesophase, it is hard to conceive how an integral membrane protein would end up remaining in the aqueous solution following the cubicon method. Provided that the protein solution and the lipid or mesophase are completely homogenized, partitioning should take place and the phase-separated aqueous solution should be devoid of protein. However, protein may be detected in the aqueous solution (really a dispersion) if the protein-laden mesophase has not been completely separated from it. By and large, this is a relatively straightforward process, provided that the protocol, as described above and as illustrated in supplementary Video 1, is followed. However, in cases in which the mesophase exists as a fine dispersion of particles, complete separation may prove elusive, with small amounts of the mesophase containing reconstituted protein remaining in the separated aqueous solution. Upon analysis, that protein will show up and be registered erroneously as not having partitioned into the mesophase. We encountered this situation with one of our reference proteins, AlgE, which had been solubilized in the detergent LDAO. We suspect that LDAO was responsible for altering the physical properties of the mesophase and for contributing to the formation of small mesophase particles. Some of these presumably were not captured in the mesophase and contaminated the separated aqueous solution. In this instance, a mere 0.15% of the protein ended up in the aqueous solution, which was inconsequential. In situations in which the mesophase exists as a fine dispersion, special care and additional time must be devoted to separating the mesophase and aqueous solution (Step 12). Reducing the detergent concentration in the protein solution to its lowest acceptable level is also recommended. Should the problem persist, an alternative detergent might be considered. In our experience, other components in the protein buffer solution do not pose problems with regard to mesophase dispersion. However, these cannot be dismissed out of hand and should be investigated in due course, should problems remain that cannot be attributed to the detergent

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mesophase was viscous, optically clear and non-birefringent, as expected for the pure cubic phase. Material so prepared is being used in ongoing CFTR crystallization trials.

DgkaDgkA is an α-helical membrane protein much used in our research as a reference for innovations in the in meso method6,23,27–30. It crystallizes at 4 °C in mesophase prepared as a 3:2 (vol/vol) mixture of 9.9 MAG and protein solution at 12 mg DgkA/ml10. The corresponding overall concentration of protein in the mesophase is 4.8 mg/ml. The solubilizing detergent is DDM.

To mimic a situation in which a protein such as CFTR cannot be concentrated above 0.5 mg/ml, DgkA at 12 mg/ml in Buffer A (buffer composition listed in table 2) was diluted with Buffer A to 0.5 mg/ml. This dilute solution was used in 12 rounds of reconstitution by the cubicon method to incrementally ramp up protein concentration in the mesophase. The initial volume of 9.9 MAG used was 20 µl and a total of 480 µl of protein solution was homogenized with it through rounds of reconstitution. In a final step, 10 µl of 9.9 MAG was added to convert the protein-enriched mesophase dispersion to the pure, optically clear non-birefringent cubic phase for use in crystallization trials.

The aqueous solution that phase-separated from the mesophase following rounds of reconstitution was checked for protein content (supplementary Fig. 8). None was detected. Assuming that all the DgkA had partitioned into the mesophase, the protein concentration in the mesophase is estimated at 4.8 mg/ml. Similarly, had all the detergent in the buffer transferred to the mesophase bilayer, its concentration post cubicon would be estimated at 3.8 mM DDM.

DgkA-laden mesophase at 4.8 mg of protein per ml is suitable for use directly in in meso crystallization trials10. Trials were set following protocols that were well established for this benchmark protein at 4 °C with a minimum of screening around optimized precipitant conditions (supplementary Methods). Crystals grew (Fig. 5a) and a complete diffraction data set was obtained with a single 50-µm-sized crystal in a cryo-stream at 100 K. The structure, solved by molecular replacement to a resolution of 3.1 Å (Fig. 5e; data collection and refinement statistics are reported in supplementary table 3), is essentially identical to that of DgkA crystallized without cubicon intervention (PDB ID 3ZE5).

peptstThis protein is an α-helical peptide transporter. It produces crystals at 20 °C by the in meso method in 9.7 MAG at a concentra-tion of 10 mg/ml in Buffer B (table 2)12. The volume ratio of lipid to protein solution used with 9.7 MAG is 1:1. In a repeat of the DgkA study just described, the starting PepTSt solution was diluted with Buffer B from 10 to 0.5 mg of protein per ml. This was used in 12 rounds of reconstitution that consumed in total 20 µl of 9.7 MAG and 400 µl of protein solution.

Assuming complete partitioning of protein into the mesophase bilayer, the concentration in the mesophase would be 5 mg PepTSt per ml, which corresponds to a 20-fold concentration in starting solution. The absence of protein in the excess aqueous solution recovered following the rounds of reconstitution (supplementary Fig. 9) supports the assumption. If all the deter-gent in the aqueous solution partitioned similarly, a final concentration of 1.8 mM DDM in the mesophase would be expected.

The resulting mesophase at 5 mg PetTSt per ml was used in crystallization trials at 20 °C. Pyramid-shaped crystals, ranging in size from 20 to 40 µm in maximum dimension, grew and were harvested after ~15 d (Fig. 5b). Data collection at 100 K yielded a full data set and a structure by molecular replacement to 2.4 Å from a single crystal (Fig. 5f). The cubicon and reference (PDB ID 4D2B) structures were very similar, with a backbone RMSD (root mean square deviation) value of 0.146 Å over 443 residues.

b2arThe first non-rhodopsin G protein-coupled receptor (GPCR) structure was obtained using β2AR crystals produced by the in meso method11,31. β2AR was also the GPCR partner in a complex with a G protein whose landmark structure was solved, again using in meso-grown crystals21. Crystals were grown in mesophase prepared with 9.9 MAG or 7.7 MAG doped with 10% (wt/wt) cholesterol, respectively. These are the same mesophase compositions we use with CFTR. Accordingly, β2AR crystallization in 9.9 MAG/cholesterol was a good benchmark with which to perform an evaluation of the cubicon method.

β2AR, as a fusion construct with T4-lysozyme in complex with the inverse agonist carazolol, crystallizes by the in meso method at 20 °C11. Typically, a solution at 45 mg of protein per ml in Buffer C (table 2) together with lipid (9.9 MAG containing 10% (wt/wt) cholesterol) in a 2:3 volume ratio is used to generate mesophase at 18 mg of protein per ml for crystallization. To evaluate the cubicon method with β2AR, the protein solution diluted with Buffer C to 1 mg/ml was used in 12 rounds of reconstitution. As no protein was detected in the excess aqueous solution post reconstitution (supplementary Fig. 10), the mesophase had an estimated concentration of 18 mg/ml, corresponding to a cubicon concentration factor of 45-fold. A final LMNG concentration of 1.5 mM in the mesophase is expected should all the detergent in the aqueous solution end up in the mesophase.

In meso crystallization trials were set up using the β2AR-laden mesophase at 20 °C. Lens-shaped crystals, 30 µm long and 5 µm in maximum thickness, were obtained and were harvested after 15 d (Fig. 5c). A single crystal at 100 K was sufficient to obtain a complete data set and a structure solved by molecular replacement to 3.2 Å resolution (Fig. 5g). The model (PDB ID 2RH1) and cubicon structures of β2AR are essentially identical, with a backbone RMSD value of 0.066 Å.

http://www.rcsb.org/pdb/explore/explore.do?structureId=3ZE5

http://www.rcsb.org/pdb/explore/explore.do?structureId=4D2B

http://www.rcsb.org/pdb/explore/explore.do?structureId=2RH1

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algeTo explore its more general applicability, the cubicon method was tested with a β-barrel protein, the alginate transporter AlgE, dissolved in the zwitterionic detergent, LDAO. AlgE crystallizes by the in meso method at 20 °C in 9.9 MAG at a protein concentration of 8 mg/ml in the mesophase, corresponding to a starting protein solution concentration of 20 mg/ml13. The protein solution used in the cubicon trial had been diluted with Buffer D (table 2) to 1 mg of AlgE per ml. Ten rounds of reconstitution raised protein and detergent concentrations in the mesophase to estimated values of 8 mg/ml and 13.1 mM, respectively. This was then used to set up in meso crystallization trials, which generated crystals and a structure from a single 5 × 75 µm2 crystal (Fig. 5d) to a resolution of 2.7 Å (Fig. 5h). Reference (PDB ID 4AFK) and cubicon structures of AlgE are essentially identical.

With DgkA, PepTSt and β2AR, the protein-laden mesophase separated easily as a sticky mass from excess, protein-depleted aqueous phase during the cubicon process. With AlgE, by contrast, the mesophase existed more as a dispersion of particles that required time and effort to separate from excess aqueous solution following individual rounds of reconstitution. A small amount of the particulate material was removed with the aqueous solution between reconstitution rounds, which accounts for the trace protein detected therein (supplementary Fig. 11). The loss is estimated at <0.2% of the total protein, which, as expected, had an imperceptible effect on crystallization outcome.

A summary of the results for the four reference proteins is provided in table 3. All cubicon structures are highly similar to those solved previously by conventional reconstitution using the same construct and lipid/detergent system (RMSD values between 0.07 and 0.23 Å).

The cubicon method is a simple, effective and generally applicable means for raising membrane protein concentrations in the lipid bilayer of the cubic phase to a level suitable for direct use in in meso crystallogenesis. We demonstrated that the method works with a range of membrane protein types and sizes, and with different solubilizing detergents and host lipid types and compositions. Furthermore, the cubicon method can be used for any downstream application for which membrane proteins reconstituted in a lipid membrane at high concentrations are required, including functional and biophysical characterization studies, ligand screening, drug delivery, antibody production and complex formation.

accession codesAll diffraction data and refined models have been deposited in the Protein Data Bank: 5D6I (DgkA), 5D6K (PepTSt), 5D6L (β2AR), 5IYU (AlgE).

50 µm

Cytoplasm

Cytoplasm Cytoplasm Periplasm

50 µm 30 µm 50 µm

a

e f g h

b c d

Figure 5 | Structures solved based on crystals grown using protein-laden mesophase generated by the cubicon method. (a–d) Crystals of DgkA in 9.9 MAG (a), PepTSt in 9.7 MAG (b), β2AR in 9.9 MAG doped with 10% (wt/wt) cholesterol (c), and AlgE in 9.9 MAG (d). (e–h) Structures of DgkA at 3.1 Å (e), PepTSt at 2.4 Å (f), β2AR at 3.2 Å (g) and AlgE at 2.7 Å (h) are shown in cartoon representation. Carazolol in g is represented as spheres (green). Lipid and detergent molecules were removed for clarity. Approximate locations of the membrane boundaries are indicated by horizontal black lines.

https://www.rcsb.org/pdb/explore/explore.do?structureId=4AFK

http://www.rcsb.org/pdb/explore/explore.do?structureId=5D6I

http://www.rcsb.org/pdb/explore/explore.do?structureId=5D6K

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http://www.rcsb.org/pdb/explore/explore.do?structureId=5IYU

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Note: Any Supplementary Information and Source Data files are available in the online version of the paper.

acknowleDGMents The authors thank past and current members of the Membrane Structural and Functional Biology Group for their many helpful contributions. The work was funded by Science Foundation Ireland (12/IA/1255) and the Cystic Fibrosis Foundation. P.M. was supported by a grant (WELBIO CR-2012S-04) to C. Govaerts (Université libre de Bruxelles) from the Belgian National Funds for Scientific Research. The support from beamline scientists at the Swiss Light Source, Villigen, Switzerland and the Diamond Light Source, Didcot, UK, is gratefully acknowledged.

autHor contrIbutIons P.M. concentrated CFTR; produced and purified DgkA and PepTSt; concentrated, analyzed, crystallized, collected and processed diffraction data for DgkA, PepTSt and β2AR, and SAXS data for DDM, LMNG and DMNG; and solved, refined and analyzed the structures of DgkA, PepTSt and β2AR. D.W. produced and purified AlgE and concentrated, analyzed, crystallized and solved its structure; refined and analyzed the structure of AlgE; and prepared samples and analyzed SAXS data for LDAO and for the mesophase samples of the reference proteins that were used for crystallization. L.A.A. and T.J.J. produced and purified CFTR. X.L. produced and purified β2AR. M.C. designed the research and J.R.R., B.K.K. and M.C. provided project supervision. M.C. wrote the manuscript with input from D.W. and P.M.

coMpetInG FInancIal Interests The authors declare no competing financial interests.

Reprints and permissions information is available online at http://www.nature.com/reprints/index.html. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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The cubicon method for concentrating membrane proteins in ... · nature protocols || VOL.12 NO.9 2017 | 1745 IntroDuctIon The primary application of the lipid cubic phase or in meso