The CRACAM Robot: Two-Dimensional Crystallization of Membrane Protein

Abstract

Membrane proteins are key cellular components that perform essential functions. They are major therapeutic targets. Electron crystallography can provide structural experimental information at atomic scale for membrane proteins forming two-dimensional (2D) crystals. There are two different methods to produce 2D crystals of membrane proteins. (1) either directly in the bulk of the solution (2) or under a lipid monolayer at the air-water interface. This extra lipid monolayer helps to pre-orient the proteins in order to facilitate the growth of 2D crystals. We present here these two methods for 2D crystallization of membrane proteins implemented in a fully automated robot called CRACAM. These methods require small volume of low concentration of proteins, making it possible to explore more conditions with the same amount of protein. These automated methods outperform traditional 2D crystallization approaches in terms of accuracy, flexibility, and throughput.

1 Introduction

Membrane proteins are essential for cellular life; therefore, they are major targets for drugs design. The atomic structure of a membrane protein is a prerequisite for understanding its function. The rapid method development in X-Ray crystallography has led to a massive increase of experimentally solved structures, namely 125,463 in 2016 [1]. However, structural information on membrane proteins is lagging behind because it is difficult to growth three-dimensional (3D) crystals. The number of unique membrane protein structures available is, as of today, 667 [2]. Moreover, proteins in 3D crystals are usually not in their native lipidic environment, which may prevent their native oligomeric state or conformations. Membrane protein electron crystallography was pioneered in the 1970s by Henderson and Unwin through their studies of bacteriorhodopsin [3] and relies on cryo-electron microscopy (cryo-EM) of two-dimensional (2D) crystalline specimens of membrane proteins in a lipid bilayer. This method is thus ideal for studying the structure of membrane proteins in their natural membrane environment.

Due to their hydrophobic nature, membrane proteins have to be kept in solution with detergent during purification . Then 2D crystals are typically grown by reconstitution of purified, detergent-solubilized membrane proteins into lipid bilayers at a high enough density to favor the formation of a regular array.

There are two different methods to produce 2D crystals of membrane proteins. Either directly in the bulk of the solution or just underneath a lipid monolayer, this extra lipid monolayer helping to pre-orient the proteins in order to facilitate the growth of 2D crystals. In both cases, 2D crystal growth is achieved by removing the detergent from ternary mixtures consisting of detergent micelles, solubilized proteins, and solubilized lipid molecules or from detergent-destabilized lipid vesicles. Several methods for detergent removal have been described such as (1) dialysis against detergent-free buffer [4], (2) adsorption of detergent molecules to polystyrene beads, Bio-Beads ^® [5], (3) dilution of the corresponding mixture below the critical micellar concentration (CMC) of the detergent [6, 7], (4) the use of cyclodextrin to chelate the detergent in solution [8].

Concerning the crystallization in the bulk of the solution, lipids, detergent, and proteins are mixed in a solution, then the detergent is removed using one of the methods described above (Fig. 1). As for the crystallization under a lipid monolayer, the protein is primarily pre-oriented on a lipid monolayer, then the detergent is removed and the protein reconstituted in a lipid bilayer to form 2D crystals (Fig. 1). Note that with this latter 2D crystallization method, the geometry is more complex. Membrane proteins are reconstituted in lipid bilayer just below the lipid monolayer. In other words, we have, at the air-water interface, the lipid monolayer and attached to it, just below, the single layer of 2D crystal. Crystallization on a lipid monolayer is an elegant method because it makes possible to work with very dilute protein solutions and still generate locally high concentration of protein constrained in 2D. Nonetheless, the proteins retain sufficient mobility to allow for organization into crystalline 2D arrays by lateral diffusion. Lipid monolayers can be spread, driven by surface tension, over the whole air-water interface of a drop to form a flat, one molecule thick film. This provides a substrate for protein binding, leading to a layer of closely packed proteins at the interface which can be organized into a 2D crystal suitable for structure determination by electron crystallography .

Fig. 1

Methods for the growth of 2D crystals of membrane proteins. Method for the growth of 2D crystals in the bulk of the solution: 2D crystals are grown by reconstitution of purified, detergent-solubilized membrane proteins into lipid bilayers at a high enough density to favor the formation of a regular array, the detergent is removed using cyclodextrin. Method for the growth of the 2D crystals on a lipid monolayer: After the lipid working solution (ligand –lipid mixed with diluting lipid) has been deposited at the top of a buffer drop and concentrated solution of the membrane protein and mixed detergent micelles and reconstituted lipids has been injected through the side tube to the bottom of the well. Over the next 24 h the membrane protein in the detergent binds to the ligand-lipid at the interface. The detergent is removed by the addition of cyclodextrin through the injection side at the side well and growth of 2D crystals takes place

Identifying the conditions for growing 2D crystals requires screening over a wide range of factors including pH, temperature, lipid composition, lipid-to-protein ratio, detergent, amphiphiles, mono- and divalent-ions, inhibitors, and ligands . A systematic screen over all these factors generates large combinations of possible reagent, which should ideally be sampled to cover the majority of 2D crystallization conditions [9].

In the past various systems of 2D crystallization of membrane proteins were described [10,11,12]. For the first time our pioneering robotic system CRACAM (Chain Robotic for the Analysis and the 2D CrystAllization of Membrane proteins) allows not only the crystallization of membrane protein in the bulk of a solution but also the 2D crystallization on a lipid monolayer. Our newly developed robot CRACAM is using cyclodextrin to chelate the detergent in solution. Cyclodextrin is cyclic oligosaccharides, composed of five or more of a-D-glucopyranoside units, with a hydrophobic interior. They form inclusion complexes with detergents or mixture of detergents regardless of their CMC s. Because cyclodextrin-detergent complexes form at a specific stoichiometry, detergent can be removed in a precisely controlled manner [8].

2 Materials

2.1 The Robot Design

We have customized the basic platform from Hamilton: MICROLAB^® STAR Line.

This platform for liquid handling is equipped with four pipetting channels (Fig. 2, number 5). There are numerous advantages working with this platform, the pipetting channels can pipette volumes as low as 0.5 μL and as high as 1000 μL. The Dynamic Liquid Classification tool enables the system to classify liquids and therefore, adapt the pipetting parameters according to the liquid used. This is particularly useful when using aqueous and nonaqueous solutions (chloroform or hexane) and often small volumes. This tool increases reliability and reduces variability . Another interesting feature is the liquid level detection. The transport of the plates on the deck is performed using a CO-RE Gripper that can be picked up by two single channels in parallel during a run. This prevents the need for a robotic hand. We have customized the deck of the platform in order to meet our specifications (Fig. 2).

Fig. 2

Customized crystallization deck for CRACAM. 1: Mother solutions rack: protein, lipid, detergent, buffer solutions; 2a: Eppendorf or PCR tubes rack for the 2D crystallization in solution; 2b: Rack for 2D crystallization on a lipid monolayer. See also Fig. 3; 3: Heating and cooling block from Inheco GmbH; 4: Stirrer plate for homogenization of the solution; 5: Four channels pipetting; 6: Tips (10, 50, 300, and 1000 μL); 7: Computer; photo credit: © IMPMC - Cécile Duflot

The working solutions (lipids, proteins, buffer) are placed on the rack (Fig. 2, position 1). Crystallization in solution takes place in eppendorf or PCR tubes of various size allowing crystallization in small amount such as 10 μL up to 1.5 mL. We implemented the system using 96 SBS type plates to increase the number of conditions to screen (Fig. 2, position 2a). 2D crystallization on a lipid monolayer is taking place on dedicated crystallization trough which are fitted into aluminum SBS-size plates (Fig. 2 position 2b). These troughs had to meet strict specifications, which are described in Subheading 2.2. An appropriate heating and cooling block (Inheco) regulates the temperature (Fig. 2, position 3) and the homogeneity of the solutions is performed individually for each well with a magnetic stirrer (Fig. 2, position 4) and microscopic magnetic sphere (NdFeB magnetic sphere from EarthMag GmbH) placed at the bottom of each crystallization well. The aluminum SBS-size plates are tightly stackable in order to prevent the evaporation of the solution (Fig. 3b).

Fig. 3

Customized setup for the crystallization on a lipid monolayer. (a) Aluminum plates SBS size compatible with space for 24 crystallization troughs and 15 water reservoirs, these reservoirs are filled with water in order to keep a high level of humidity and to avoid any evaporation of the crystallization solution. (b) The plates are stackable, a hermetic seal is used between each stacked plate, in order to keep a high level of humidity and avoid any evaporation of the crystallization solution. (c) Teflon^® crystallization through is made of the crystallization well where the crystallization takes place and the injection well where the various solutions of proteins, lipids, detergent or cyclodextrin are injected. Photo credit: © IMPMC - Cécile Duflot

The robot is controlled through the software Venus3. This software possesses two main components. The first “experiments layout on the deck” allows recreating a virtual 3D version of the deck. To do so, a large database provided allows the user to select a whole range of objects (tubes, plates, cones, liquid, racks…). The second component “experiment monitoring” is dedicated solely to programming, and creates a sequence of commands for manipulating and initiates the protocol. The parameters to conduct the experiment are set up at the beginning of the experiments: initial volumes for the various solutions according to the final desired lipid/protein ratio cyclodextrin to add, temperature profile, homogenization periods , and frequency.

2.2 The Crystallization Trough for the 2D Crystallization on a Lipid Monolayer

The Teflon^® crystallization trough is made of a crystallization well (3.5 mm diameter, 32 μL) and the injection well, much smaller (1.5 mm diameter) (Fig. 3c), used to inject the solution (proteins, lipids, buffer, cyclodextrin) once the monolayer has been formed at the air:water interface in the crystallization well. This helps to keep the lipid monolayer intact. These crystallization troughs are fitted into aluminum SBS-size plates. The plates have been “customized” in order to be stackable and have the further 15 water wells in order to control evaporation during crystallization (Fig. 3b).

2.3 Preparation of the Solution

Prepare all the solutions using ultrapure water and analytical grade agents. Sodium azide (0.01%) in the buffer and protein solution is required to prevent bacteria to growth.

2.3.1 The Solutions for the Crystallization in the Bulk of the Solution

• 1. Lipids for the protein reconstitution (reconstituting lipids): these lipids will form bilayers in which the proteins will insert and hopefully organize in 2D crystals. Usually, the reconstituting lipids are Escherichia coli polar extract, Soybean polar extract, DMPC (1,2-dimirystoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauryl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), POPA (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and DOPA (1,2-dioleoyl-sn-glycero-3-phosphate).

  These lipids should be added in the solution in the form of mixed micelles with detergent. Many different detergents can be used. The detergent can be the same as used for the solubilization of the membrane protein or different such as TritonX100 ; n-dodecyl-β-d-maltopyranoside (DDM ); decyl β-d-maltopyranoside (DM); 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) ; 1-O-n-Octyl-β-d-glucopyranoside (OBG).

  These lipids can also be added in the solution in the form of preformed liposomes .

• 2. Various solutions containing the concentrated solubilized membrane protein (with which it is hoped to form 2D crystals).

• 3. Buffer solution for the dilution of the protein in detergent so the required lipid/protein ratio is obtained after addition of lipids. The lipid to protein ratio ranged from 0.2 to 1.4 (w/w).

• 4. Cyclodextrin solution should be prepared in water at the appropriate concentration.

2.3.2 The Solutions for the Crystallization on a Lipid Monolayer

• 1. The solutions 1, 2, 3, and 4 will be necessary. In addition, another solution containing the lipid forming the monolayer at the air/water interface is also needed.

• 2. Lipids forming the monolayer: regular (non-fluorinated) lipids or fluorinated lipids [13] bearing a Ni²⁺-NTA group for interaction with His-tagged proteins, or any other lipid–ligand conjugate for the interaction with the corresponding receptor. These lipids are different from the lipids that form the lipidic bilayers where the protein will insert to form the 2D crystals (see Notes 1 – 3 ).

2.3.3 Transfer to an Electron Microscope Grid and Observation

• 1. Electron microscope grids (400 mesh) covered with a carbon film.

• 2. Electron Microscope.

  These are necessary for the observation of the 2D crystals under the electron microscope.

3 Methods

• 1. Lipids forming the lipid monolayer: the lipids may be proned to oxidation, and therefore, are best stored as a dry powder under an argon atmosphere at −20 °C, in an air-tight glass container. A mother solution (5 mg/mL) is produced by solubilizing the lipids in an organic solvent (chloroform:hexane 1:1 v/v). This lipid solution is best stored also at −20 °C under argon in a glass container with a Teflon^® cap. The working solution is made usually of the ligand -lipid and diluting-lipid at a molar ratio between 1:10 and 1:3 at a concentration of 500 μM. The ligand-lipid is the lipid which may specifically interact with the protein, and the dilution-lipid will act as a fluid matrix. When fluorinated lipids are used, ligand- and diluting-lipids must be fluorinated otherwise, segregation between fluorinated and non-fluorinated lipids can appear.

• 2. Lipids for the protein reconstitution (reconstituting lipid).

  These lipids can be solubilized in detergent. Aliquots of 5 mg of phospholipids DOPC, POPC , E. coli lipids, or other lipids (see Subheading 3.1 of Chapter 2) should be prepared and stored as a dry powder under an argon atmosphere at −20 °C in an air-tight glass container. The detergent may be supplied as a powder or as concentrated solution in water, or solubilized in a solvent (this is the case for DHPC ). When needed, one aliquot of 5 mg lipid is solubilized in 1 mL deionized water with detergent, so the final concentration of lipid is 5 mg/mL. If the detergent is solubilized in a solvent, the solvent should be thoroughly evaporated. Aliquots of dried lipid and detergent can be kept in the same air-tight glass until addition of 1 mL deionized water or buffer for complete solubilization of the lipid in the detergent. The final concentration of the detergent should be at least ten times its cmc. A clear indication of the complete solubilization of the lipid in the detergent is the limpidity of the lipid detergent solution: this preparation should be perfectly clear. Depending on the properties of the reconstituting lipid and the detergent, the solubilization of lipid in the detergent could be instantaneous or may require stirring overnight at room temperature, or at higher temperature (up to 45 °C), or sonication for 15 min (in a sonicator bath taking care that the lipid solution does not heat up too much). If the solution is still opaque after the above treatment, it might be useful to add more detergent, or apply a more intensive sonication treatment. Solubilized lipid in the detergent can be kept for up to a month at 4 °C under an argon atmosphere, in an air-tight container.

  The added lipids for the reconstitution of the protein can also be added as liposomes. The protocol is the same as above but without addition of detergent. When needed, one aliquot of dried 5 mg lipid is solubilized in 1 mL deionized water and sonicated for about 15 min, the solution keeps turbid. The sonication should be always performed just before the 2D crystallization experiments.

• 3. Protein solution should be at a concentration about 1.0 mg/mL ideally.

• 4. Cyclodextrin solution should be solubilized in water at a concentration ranging between 0.5 and 10 mg/mL depending on the final concentration needed.

• 5. Protocol for the crystallization in bulk solution`

  • (a) The buffer, membrane protein solution, lipid solution for the reconstitution of the membrane protein and cyclodextrin are placed on the rack at position 1 Fig. 2 on the deck of the robot and kept at 4 °C (It is worth considering adding some azide (0.01%) in the buffer and protein solutions to prevent bacteria to growth). The crystallization tubes are in position 2a. The parameters to conduct the experiment are set up at the beginning of the experiments as described in Subheading 2.1 in chapter 2. Then the robot follows this sequence using the pipetting channels.

  • (b) Buffer (position 1 on the deck, Fig. 2) is deposited in the crystallization tube.

  • (c) The solution containing the concentrated membrane protein in the detergent and the lipids (as mixed micelles in detergent or just solubilized in water, see step 1) is then injected (one after the other) in the crystallization tube. The final concentration of the membrane protein should be between 0.5 and 1.5 mg/mL and the reconstituting lipid concentration should be such as the lipid/protein ratio is in range 0.2–1.4 (w/w). The total volume is variable from 10 μL up to 1.5 mL.

  • (d) Good homogeneity is ensured by using the magnetic stirring beads placed at the bottom of the tube. The crystallization plates are transported using a CO-RE Gripper on the magnetic stirrer.

  • (e) The plates are then transported to the cooling block (Fig. 2 position3) and the reconstitution step of the membrane protein into the lipid bilayer is realized by elimination of detergent using addition of cyclodextrin. This step of reconstitution can be spread over several days. The amount of cyclodextrin to add is important. The most probable complexes between cyclodextrin and the detergents are showing a ratio between 1 and 2 [8]. The cavity of a cyclodextri n molecule is about 8 Å deep, offering accommodation for a C8 chain [8]. Therefore, the total amount of cyclodextrin to add should be between one to twice the amount of detergent present in the solution (see Note 4 ).

  • (f) The specimen is then ready to be transferred on an electron microscope grid for observation.

• 6. Protocol for the crystallization on a lipid monolayer.

  • (a) The Teflon^® crystallization troughs should be thoroughly cleaned with a detergent, boiled in water for 10 min or sonicated in chloroform for 5 min, followed by extensive rinsing with deionized water. The Teflon^® surface should be hydrophobic after this treatment.

  • (b) The buffer, proteins, lipid solution for reconstitution in lipid bilayer, lipid in chloroform/hexane for the lipid monolayer and cyclodextrin are placed on the rack at position 1, Fig. 2, on the deck of the robot and kept at 4 °C (It is worth considering adding some azide (0.01%) in the buffer and protein solutions to prevent bacteria to growth.) The crystallization throughs are in position 2b. The parameters to conduct the experiment are set up at the beginning of the experiments as described in Subheading 2.1. Then the robot follows this sequence using the pipetting channel.

  • (c) Buffer is placed in the crystallization trough well.

  • (d) The lipid working solution, for the monolayer at the air:water interface (regular or fluorinated ligand -lipid, 1 μL at 500 μM), is deposited at the top of the crystallization drop. The lipid spreads and leaves a film at the air-water interface while the organic solvent evaporates. The amount of lipid is more than would be required for a single layer, usually 5–10 times surplus. The excess lipid forms a reservoir at the edge of the Teflon^® well.

  • (e) The solutions containing the concentrated membrane protein in detergent and the lipids (as mixed micelles in detergent or just solubilized in water see Subheading 2.1) are then injected, one after the other, through the injection well. The final concentration of the membrane protein in the well should be between 50 and 150 μg/mL and the reconstituting lipid concentration should be such as the lipid-to-protein ratio is in range 0.2–1.4 (w/w), the total volume in the crystallization well is 32 μL.

  • (f) Good homogeneity is ensured by using the magnetic stirring beads placed at the bottom of the tube. The crystallization plates are transported using a CO-RE Gripper on the magnetic stirrer.

  • (g) The plate is then transported to the cooling block (Fig. 2 position3) stacked up in order to avoid any evaporation. During this step, which should last up to 24 h, the protein in the detergent should bind to the ligand–lipid at the interface. At this stage, a screen can be done, transferring the layer on an electron microscope grid in order to find out which lipid and which lipid/protein ratio give the best result (the higher protein coverage).

  • (h) The reconstitution step of the membrane protein into the lipid bilayer is realized by injection of cyclodextrin into the bottom of the well. The addition of the cyclodextrin should be performed when binding of the membrane protein to the lipid monolayer has reached completion (high protein density on the lipid monolayer). The total amount of cyclodextrin to add should be between one to twice the amount of detergent present in the solution (see Note 4 ).

  • (i) Usually after 24 h, most part of the detergent in solution has been trapped on the cyclodextrin and the protein bound to the lipid film at the air-water interface is reconstituted into a lipid bilayer (reconstituting lipid) and hopefully forms 2D crystals. The specimen is ready to be transferred on an electron microscope grid for observation.

• 7. Transfer on grids and staining procedure for observation with an electron microscope .

  • (a) Crystallization in the bulk:

    3 μL of the crystallization solution is pipetted and deposited on a hydrophilic grid, the specimen on the grid is rinsed by touching a drop of water (several cycles of water rinsing are sometimes necessary particularly when cyclodextrin is used), stained by a drop of 1–2% uranyl acetate. The excess of stain is blotted with a filter paper, and the grid is air dried.

  • (b) Crystallization on a lipid monolayer:

    Transfer of 2D crystals onto a microscope grid. The 2D crystals or protein layers are transferred to the electron microscope grid through hydrophobic contacts between the lipid chains of the ligand -lipid and the carbon film on the grids. The grid is deposited on the top of the crystallization drop, left for about 1 min and then withdrawn and prepared for observation under the microscope. The transfer often works best with grids covered with a very hydrophobic carbon film. The film can be rendered hydrophobic through baking for 1 h at 150 °C. In order to visualize the 2D crystals, the specimen on the grid is rinsed by touching a drop of water (several cycles of water rinsing are sometimes necessary particularly when cyclodextrin is used), stained by a drop of 1–2% uranyl acetate. The excess of stain is blotted with a filter paper, and the grid is air dried (see Note 5 ). Alternatively, the specimen can be prepared for cryo-electron microscopy observation. In that case, after transfer the grid is blotted for 5–8 s with a filter paper and plunged into liquid ethane. The sample is then preserved in a frozen-hydrated state.

    An automatic staining procedure is in development on the robot, we are using a suction system in order to move the grids from the specimen crystallization through to the staining drops.

4 Notes

• 1. Some requirements for 2D crystallization of proteins on a lipid monolayer:

  • Limiting the protein to a plane.

  • A high concentration of the protein in a plane.

  • Orientation of the protein.

  • Providing mobility of the protein within the plane to allow sampling of various interactions arrangements.

This last point can be of help by the physical properties of the monolayer:

The physical properties of the monolayer system are determined mainly by the chemical composition of the lipids, the temperature and the composition of the underlying buffer. In order to allow protein crystallization, these parameters have to permit the lateral diffusion of the protein molecules attached to the monolayer. To achieve a favorable physical state of the lipid layer, it has often proven useful or even essential to use mixtures of different lipids. As these additional lipids usually do not carry a functional group, they are generally referred to as diluting lipids. A reason for the dependence on dilution lipids might be the difference in surface covered by proteins and the much smaller lipids. A phospholipid occupies 50 times less area than a 100 kDa globular protein. Therefore, one protein molecule can interact with many lipid molecules. This makes it clear why the composition of the lipid layer is of high importance. Furthermore, it is possible to adjust the fluidity properties for a given monolayer by mixing functionalized lipids with diluting lipids of different structures.

Therefore various ratio of ligand-lipid to diluting-lipid for the lipid monolayer at the air water interface should be tested carefully. It is worth trying a range from 1:0 to 1:10. This ratio can have a great influence on the density at which the protein binds to the monolayer.

• 2. Adsorption through specific and not specific interaction.

  • Electrostatic interaction:

    Lipids can contain headgroups with positive, negative, or neutral charges. The attraction of opposite electrical charges provides the basis for electrostatic interactions. Proteins carry positive and negative charges, according to the acidic and basic chains of each amino acid. At acidic pH values, the protein shows a cationic behavior. In contrast, at basic pH the protein is rather anionic. Therefore, the net charge of a protein is dependent on the pH of the surrounding buffer and the number of exposed charged amino acids on the surface of the protein. The overall charge of a protein is described by a pI value. If the pH of the buffer is at the pI, the net charge of the protein is zero and therefore the capacity of the protein for electrostatic interactions is low, unless there is an unequal distribution of charged amino acids on the surface of the protein, which leads to regions with positive or negative charges. The electrostatic interaction needs to be investigated testing various pH, this might be a tedious task; therefore, it is advised to use Ni-phospholipids that will bind tightly to the His-Tag from the expressed protein.

• 3. The detergent in the bulk of the solution might solubiliz e the lipid monolayer at the interface; therefore in some case fluorinated lipids were used as described in recent papers and book chapter [13, 14]

• 4. The capability of cyclodextrin to complex any kind of detergent molecule, independently of the CMC , is a crucial advantage over the dialysis method. To perform reconstitution with cyclodextrin accurately, a precise evaluation of the amount of cyclodextrin needed to remove all the detergent from a solution is required. Ideally, detergent concentration after protein purification should be systematically measured indeed, during purification, as the solubilized protein passes through ion-exchange or affinity columns or undergoes concentration by centrifugation , the detergent in the mixture gets diluted or concentrated. Although quite a few methods exist for the determination of detergent concentrations, they are impractical for many routine applications. A homemade device [15] was proposed to measure accurately and easily the detergent concentration of any solution (detergent solution, binary or ternary mixtures). This “DropBox”method is based on the contact angle made by a sample droplet on a hydrophobic surface. The observed angle is compared with a calibration curve for the particular detergent to determine its concentration. This method was therefore used to measure the cyclodextrin e–detergent molecular ratios after cyclodextrin addition to detergent solutions. When this method is not available, the value of one to two cyclodextrins per detergent molecule (the exact ratio depends on the detergent molecule) seems to be a good approximation.

• 5. To avoid disruption of the 2D crystals during the transfer, glutaraldehyde (1 μL of a 0.5% aqueous solution), a protein cross-linker, can be added through the injection well just before placing the microscope grid. Glutaraldehyde strengthens the protein arrays [16].