Parameters for the Two-Dimensional Crystallization of the Membrane Protein Microsomal Glutathione Transferase

Parameters for the Two-Dimensional Crystallization of the Membrane Protein Microsomal Glutathione Transferase

JOURNAL OF STRUCTURAL BIOLOGY ARTICLE NO. SB984018 123, 87–96 (1998) Parameters for the Two-Dimensional Crystallization of the Membrane Protein Micr...

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123, 87–96 (1998)

Parameters for the Two-Dimensional Crystallization of the Membrane Protein Microsomal Glutathione Transferase Ingeborg Schmidt-Krey, Gerd Lundqvist,* Ralf Morgenstern,* and Hans Hebert Center for Structural Biochemistry, Department of Biosciences, Karolinska Institutet, S-141 57 Huddinge, Sweden; and *Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 210, S-171 77 Stockholm, Sweden Received April 27, 1998, and in revised form July 9, 1998

choline receptor (Unwin, 1993), Ca12-ATPase (Shi et al., 1995), and aquaporin-1 (Walz et al., 1997; Cheng et al., 1997; Li et al., 1997) are based on well-ordered, two-dimensional crystals suitable for high-resolution electron microscopy. However, as yet little is known about the driving forces of two-dimensional crystallization, and a systematic approach to the screening of crystallization parameters is not available at this point, most likely due to the fact that this method has just reached a point in its development where its use is becoming more widely applied and recognized (reviews: Ford et al., 1990; Engel et al., 1992; Jap et al., 1992; Ku¨hlbrandt, 1992; Hasler et al., 1998; Hebert, 1998). Rat liver microsomal glutathione transferase is a membrane protein with 95% sequence identity compared with the human enzyme and consists of three identical subunits with a combined molecular weight of 51,000. Its principal function is the conjugation of glutathione to a large number of hydrophobic electrophiles (Chasseaud, 1979) as well as the important property of protecting membranes against lipid peroxidation (Mosialou and Morgenstern, 1989; Mosialou et al., 1995). The microsomal glutathione transferase has three to four predicted membrane spanning regions, with most of the protein buried within the membrane bilayer and small hydrophilic domains. Structural information is crucial to find answers to biochemical questions relating to the understanding of the biochemical data available thus far, for instance, membrane topology as well as possible conformational changes that affect, or are intrinsic to, activity. The effects of various parameters were investigated to attain two-dimensional crystals of the microsomal glutathione transferase by reconstitution, to improve these crystals, and to add to the understanding of the dynamics of two-dimensional crystallization in general that might be of help in future crystallization trials of other membrane proteins. The tests of crystallization parameters could be

Various crystallization parameters were investigated to obtain two-dimensional crystals of the detoxification enzyme microsomal glutathione transferase for structural analysis by electron crystallography. The protein was crystallized by reconstitution of the solubilized trimer into proteoliposomes. Crystallization occurs when minimal amounts of lipid in the range of three lipid molecules per protein trimer are added to the dialysate. Once crystals were obtained, the effect of several parameters on the crystallization was determined. The temperature and initial detergent concentration were found to be crucial parameters in influencing the size of the crystals, and conclusions could be drawn about the rate dependence of the crystallization process. Two highly ordered crystal forms, which are suitable for structural analysis by electron crystallography, were obtained under the two-dimensional crystallization conditions described here. r 1998 Academic Press

Key Words: two-dimensional crystallization; electron crystallography; membrane proteins; microsomal glutathione transferase


Electron crystallography has become an alternative to X-ray crystallography for the structure determination of membrane proteins, which have proven to be difficult to crystallize in three dimensions. In recent years, after the developmental work of Henderson and co-workers (Henderson and Unwin, 1975; Unwin and Henderson, 1975; Baldwin and Henderson, 1984; Henderson et al., 1986, 1990; Baldwin et al., 1988; Grigorieff et al., 1996), the structures of two membrane proteins have been solved to atomic or near-atomic resolution by electron crystallography: bacteriorhodopsin (Henderson et al., 1990; Grigorieff et al., 1996; Kimura et al., 1997) and plant light-harvesting complex II (Ku¨hlbrandt et al., 1994), and the emerging structures of the nicotinic acetyl87

1047-8477/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.



performed because the microsomal glutathione transferase forms two-dimensional crystals with a high reproducibility. MATERIALS AND METHODS Microsomal glutathione transferase was purified from rat livers according to Morgenstern et al. (1982). For the crystallization trials, the protein was stored at pH 8 in buffer A (10 mM potassium phosphate, 20% glycerol, 0.1 mM EDTA, 1 mM glutathione, 1% Triton X-100, 0.1 M KCl). Phospholipid (bovine liver phosphatidylcholine, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, or dioleoyl phosphatidylcholine) in chloroform (Avanti Polar Lipids, Inc.) was placed in a round-bottom flask, and the chloroform was evaporated under a stream of nitrogen gas. On careful evaporation, the lipid was resolubilized in detergent solution of either 1% (w/w) Triton X-100 (Boehringer) or 2:1 (w/w) cholate, followed by sonication for approximately 30 s. Various concentrations of the solubilized lipid were added to the protein in buffer A. Usually the protein was at a final concentration of 1 mg/ml for the dialysis. The protein concentration was also varied in the range 0.5–1.75 mg/ml. The dialysate was placed in the dialysis device, such as a dialysis system consisting of a capillary with membranes at both ends, which is available for various small volumes (a kind gift from Professor Alain Brisson), X-ray dialysis buttons, Spectra dialysis tubing, the Pierce Microdialyzer, and Pierce dialysis cassettes with molecular weight cutoffs in the range 10,000–14,000, and dialyzed against 0.05–2 liters of detergent-free buffer E at pH 6.5–10.2 (10 mM potassium phosphate, 0–40% glycerol, 0.1 mM EDTA, 1 mM glutathione, 0.050–1 M KCl). Even though the crystallization is highly reproducible under optimal conditions, in below 10% of the preparations crystals or membranes do not form under these conditions. Therefore, successful as well as unsuccessful crystallization conditions were repeated several times in parallel with controls. The dialysis was sampled at different time intervals, and the dialysate was negatively stained with 1% uranyl acetate on electron microscope grids. The progress of the crystallization trials was monitored with Phillips EM420 and CM120 electron microscopes. At later stages, advantage was taken of a Tietz slow-scan CCD camera, which makes it possible to examine a larger number of membranes for crystallinity. Most of the protein is buried within the membrane and thus has low contrast in negative stain. Once the crystallization was successful, and in following setups to examine crystal quality, the microsomal glutathione transferase membranes were embedded in 0.5% glucose or tannin, and electron cryomicroscopy at 2170 to 2180°C was used for a more careful examination. Data were collected on Kodak SO-163 film, and once the crystals were of sufficient quality, electron diffraction patterns were recorded to determine the crystal quality. Images were analyzed with the EM program (Hegerl, 1996), Semper (Saxton, 1996), and the MRC programs (Crowther et al., 1996) on Sun, Silicon Graphics, and DEC Alpha workstations, respectively. The protein preparation was screened by thin-layer chromatography for any copurified lipids. Prior to the thin-layer chromatography, Triton X-100 was removed from the purified protein by dialysis for 7 days at room temperature since Triton interferes with the results of thin-layer chromatography. Buffer A containing 1% Triton X-100, the dialysate from the crystal preparation, microsomal glutathione transferase in Triton X-100, and the detergent-free, purified protein were examined for lipids as well as the presence and effects of Triton X-100. The mitochondrial lipids used as standards were lysophosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and cardiolipin. The silica gel

plates were developed in ChCl3:MetOH:HAc:H2O (25:15:4:2, v/v), and the lipid was visualized with iodine vapor. RESULTS AND DISCUSSION

Phospholipids The most essential parameter in the crystallization of microsomal glutathione transferase is the number of phospholipid molecules per protein trimer. The ratio is unusually low compared with those of crystals of other membrane proteins which typically have a range of 25–400 lipids per protein (Ku¨hlbrandt, 1992). For microsomal glutathione transferase, a lipid-to-protein ratio (LPR) in the range of 10–25 results in proteoliposomes with loosely packed protein, which are pseudocrystalline (Fig. 1a), and often smaller patches of a few packed protein molecules are found in big membranes. The projection map from such areas contains only lowresolution information and gives an indication of the trimeric nature of the protein (Fig. 1b). Once the ratio is decreased to below 5 lipid molecules per trimer, the protein forms well-ordered, orthorhombic two-dimensional crystals with p21212 symmetry, a 5 91.90 Å, b 5 90.83 Å, g 5 90°, and a membrane sheet size of up to 10 µm (Table I, Fig. 2). The orthorhombic crystal type forms most reliably at a ratio around 3 lipid molecules per trimer. At this LPR another crystal type with p6 symmetry, a 5 b 5 81.8 Å, g 5

FIG. 1. (a) Lipid-to-protein ratios around 10:1 and above result in vesicles with loosely packed microsomal glutathione transferase. The vesicle was negatively stained with 1% uranyl acetate. Scale bar 5 500 Å. (b) Correlation averaged projection structure with imposed p3 symmetry of negatively stained microsomal glutathione transferase vesicles reconstituted by dialysis with an LPR of 10. Scale bar 5 20 Å.


TABLE I Effect of the Lipid-to-Protein Ratio (LPR) on the Crystallization of Microsomal Glutathione Transferase LPR

Symmetry Unit cell




5 to #25

p21212 a 5 91.90 Å b 5 90.83 Å g 5 90° 3 Åa

p6 a 5 b 5 81.8 Å

— (loosely packed) —

g 5 120° 3 Åa

a Minimum resolution by electron diffraction of an untilted specimen.

120°, is found as well, and appears to be favored at only fractional changes below a ratio of 3 (Fig. 3). These membranes are usually somewhat smaller, with an average membrane size of 1.5 to 2.5 µm, and are equally well ordered compared with the orthorhombic crystals. Some membranes in the p6 preparations have a diameter similar to that of the big orthorhombic phospholipid sheets. Both crystal types of microsomal glutathione transferase diffract electrons beyond 3 Å (Hebert et al., 1997, Schmidt-Krey et al., submitted). The p6 membranes tend to stack at times, but sufficient single-layered membranes are present for the data collection. While the orthorhombic crystals are found in around 40% of the membranes in typical specimens, the p6 crystals often constitute more than 90% of the membranes in the p6 preparations. Since the characteristic LPRs are nearly identical, a mixture of both crystal types is difficult to avoid, even though one type is favored. The percentage of one crystal type is of less significance. By selecting the crystals for shape and identifying crystalline versus noncrystalline membranes, a success rate greater than 90% in the image and electron diffraction data can be achieved with some experience. Unfortunately this does not hold for the data collection at high tilt angles, even though long, orthorhombic vesicles can still be identified. The orthorhombic crystals are often found in membrane sheets (Fig. 2a) and somewhat extended vesicles (Fig. 4), while the hexagonal crystals tend to be round membrane sheets (Fig. 3a) or vesicles. For two-dimensional (2D) crystals of microsomal glutathione transferase, this suggests that to a certain extent the shape of the membranes is influenced by the symmetry of the crystal within the membrane or vice versa. Experiments to narrow the LPR further by fractional changes around an LPR of 3 did not show any improvements. The number of membranes merely decreased at ratios below approximately 2.2, while increased precipitation was observed. Microsomal glutathione transferase precipitates completely when no lipids are added for the dialysis.


Light-harvesting complex II does not require any lipids apart from the copurified lipids for crystallization (Ku¨hlbrandt, 1984). Similar crystal contacts were found in both crystal types of microsomal glutathione transferase (Schmidt-Krey et al., submitted). The protein was tested by thin-layer chromatography for any bound microsomal lipids which might explain why the small amount of lipid added is sufficient. Lipids were not detected in the purified, detergent-free protein preparations (not shown), while the crystal solution and the spots containing Triton X-100 showed the expected spots for lipid and Triton X-100 contamination, respectively. In an attempt to mimic the natural environment of microsomal glutathione transferase in liver, bovine liver lecithin was used for the first crystallization trials. However, using a synthetic phospholipid, such as dioleoyl phosphatidylcholine (DOPC), versus a natural phospholipid did not influence the crystallization of microsomal glutathione transferase. For bacteriorhodopsin and OmpF porin (Dorset et al., 1983; Hoenger et al., 1990) it has been demonstrated that different unit cell sizes can be obtained by varying the chain length of the lipid. For other membrane proteins this has also shown to have an effect on the symmetry (review Engel et al., 1992). Therefore phospholipids with chain lengths from C12 to C18 were tested. None of these lipids had influence on either the unit cell size as tested by electron diffraction (not shown), the symmetry of the crystals, or any other aspect of the crystallization such as the membrane size. The unit cell is so tightly packed with protein and few lipid molecules as it is that the area filled by lipid molecules might be at its minimum size without further possibility for a decrease. Phospholipase was not tested since the crystal symmetry can be controlled by the amount of phospholipid added. The LPR is so low that a decrease in the lipid content might lead to precipitation of additional protein and a reduced number of crystals. However, phospholipase might be of interest to induce crystallization in preparations that contain lipids in excess of the crystallization LPR. These findings imply that the crystallization does not depend on a specific phospholipid. However, the distances between trimers in the p6 crystal are similar to the bacteriorhodopsin p3 packing which relies on protein–lipid–protein interactions (Havelka et al., 1995), while in the orthorhombic microsomal glutathione transferase crystals protein–protein interactions could be present between trimers in opposite orientations, where closer contacts exist, as well as protein–lipid–protein interactions. These distances between microsomal glutathione transferase trimers also indicate that the p6 crystals actually contain more lipids per trimer than the initial LPR



FIG. 2. (a) Two-dimensional membrane sheet of microsomal glutathione transferase exhibiting p21212 symmetry grown by reconstitution of the solubilized protein with low lipid and high initial detergent concentrations. The membrane has been negatively stained. Similar preparations embedded in trehalose or tannin display electron diffraction beyond 3 Å. Scale bar 5 1 µm. (b) Projection map of negatively stained p21212 crystals illustrates the tight packing of microsomal glutathione transferase. The three subunits of each protein trimer can be distinguished. The unit cell (outlined) is 91.90 3 90.83 Å and contains four trimers, two of which are in the opposite membrane orientation.

used. Grigorieff et al. (1995, 1996) located the 10 lipid molecules per protein monomer in the bacteriorhodopsin model, and thus a larger number of lipids than the LPR can be anticipated in the microsomal glutathione transferase crystal packing as well due

to the distances between trimers. Possibly a minimum number of lipids that is higher than the LPR is needed to form proteoliposomes, and the remaining protein precipitates. This argument also holds for the orthorhombic crystal type since it has a



FIG. 3. (a) Hexagonal two-dimensional membrane crystals usually have a characteristic round shape. Preparations of these membranes frequently contain around 90% well-ordered membranes, but tend to stack, which especially occurs after storage for several weeks or months. The hexagonal crystals are comparable in quality to the orthorhombic crystals and diffract to about 3 Å. These membranes were negatively stained. Scale bar 5 100 nm. (b) p6 projection map of the hexagonal crystal type embedded in tannin and calculated at low resolution to illustrate the protein packing. One unit cell of a 5 b 5 81.8 Å and g 5 120° is outlined. The unit cell contains two trimers. The structure on the sixfold axis has been identified as a trimer in this position which is packed in two possible orientations (Schmidt-Krey et al., submitted).

packing density of 24.87 D/Å2 which is slightly lower than the 26.87 D/Å2 of the p6 form (Schmidt-Krey et al., submitted). Temperature Crystallization trials were compared at 4°C and room temperature (21°C). Large membranes of several micrometers were shown to form after 4–5 days of dialysis with DOPC at room temperature, while

FIG. 4. Orthorhombic crystals are usually found in membrane sheets, but can also form elongated vesicles. These membranes are as well ordered as the sheets, but are less suited for high tilt angles in the data collection for a high-resolution three-dimensional structure due to their shape and size in one direction as well as more frequent double layering of the crystal. Scale bar 5 500 nm.

only small vesicles below 0.5 µm formed in the cold room after approximately 10 days of dialysis (not shown). Triton X-100 micelles display increased attractive forces at higher temperatures (Wang and Ku¨hlbrandt, 1991). At lower temperatures the micelles might be more dispersed, whereas the Triton X-100 micelles at room temperature aggregate readily and leave the possibility for larger membranes to form on detergent removal. Increasing the temperature to 30°C during the crystallization does not have an effect on either the crystal size or the crystallinity, while these two attributes deteriorate at a dialysis temperature of around 36–38°C. A possible explanation is either a phase transition of the lipid or denaturation of the protein at elevated temperatures. Heating after or in later stages of the dialysis can lead to membrane fusion as in the case of lightharvesting complex II (Wang and Ku¨hlbrandt, 1991). In an effort to examine this possibility and to improve the order of the crystals, crystalline specimens were heated after the dialysis. Preformed orthorhombic membranes significantly deteriorated in quality after 2 to 6 h of heating at 36–38°C. After 90 min the specimens were somewhat affected in that a minor transition of hexagonal crystals to the orthorhombic type occurred. This change was so small that it was not investigated further, since the membranes can be distinguished by their characteristic appear-



ances. Incubation at room temperature after the dialysis did not improve the quality of the crystals either. Detergent The detergent composition as well as the detergent concentration was tested. For 2D crystals grown with Triton X-100 the conventional starting concentration is usually around 0.1% (Ku¨hlbrandt, 1992). This is not surprising as the low critical micelle concentration (CMC) of Triton X-100 leads to a long dialysis time. Initial trials for the microsomal glutathione transferase were carried out successfully at a starting Triton X-100 concentration of 1%. To examine the effects of other concentrations on the crystallization as well as a possible way of decreasing the crystallization time, the Triton X-100 concentration was monitored in the range 0.4–1% in increments of 0.1 (Figs. 5a, 5b). Lowering to 0.7–0.8% (Fig. 5b) does not affect the crystals. Surprisingly, detergent concentrations around 0.5–0.6% (Fig. 5a) and lower resulted in smaller, yet still well-ordered, membrane vesicles. These smaller crystals usually were significantly below 0.5 µm. Consequently, the detergent concentration was increased to 2–4% with a corresponding increase in the length of the dialysis time to 21 days (Fig. 5c). The resulting crystals were identical in appearance and quality to the crystals grown at the standard concentration of 1%. Due to the influence of the detergent concentration on crystal size, a batch crystallization, in which the specimen is simply incubated in an Eppendorf tube, is thus less likely to be developed since lower detergent concentrations are necessary for such a system. The need for high detergent concentrations also prevents a decrease in the crystallization time and gives an indication of the rate dependence of the crystal size. Another parameter that proved to be of importance for the data collection is the dialysis time. When sampling the dialysis after 5 days, large membrane sheets up to several micrometers were obtained, but crystallinity could not be observed. Well-ordered crystals form after 6 days of dialysis (Schmidt-Krey et al., 1994). However, these crystals deteriorate rapidly after approximately 7 days of storage. Increasing the dialysis time by only 2 days to 8 days provides crystals that last at least 2–4 months, and thereafter deteriorate only slowly compared with the 6-day preparations. The crystals can be stored at room temperature. The data collection and structure determination at liquid helium temperatures, with an electron microscope developed by Fujiyoshi et al. (1986, 1991), have been greatly facilitated by eliminating the necessity for frequent transports. In reconstitution experiments of microsomal glutathione transferase, Triton X-100 is re-

FIG. 5. An equally important determinant of crystal size is the detergent concentration. Concentrations below 0.6% produce small vesicles as shown in (a) for 0.4% Triton X-100. Increasing the detergent concentration to 0.7% (b) also leads to an increase in the size of membranes, which are identical to the membrane sheets grown at the optimal concentration of 1% as shown in Fig. 2a. The same results were obtained with microsomal glutathione transferase solubilized in 4% Triton X-100 (c) and dialyzed for 21 days, which means that an increase in the detergent concentration ensures formation of larger membrane sheets. Precipitation on the membranes is occasionally seen in negative stain, but is nearly absent on embedded crystals for electron cryomicroscopy. Scale bars 5 1 µm.

moved by dialysis for 7 days in the cold room (Mosialou et al., 1995). Therefore, we believe that even though it could be argued that the detergent is not completely removed after 8 days, none was visible by thin-layer chromatography (not shown)


and an insignificant amount of detergent is left at most, which is the reason for the increased longevity of the crystals. Since the durability of the crystals is directly linked to the dialysis time, the effect of additional detergent removal is the most likely explanation. A further increase in the dialysis time to 9–22 days has not been shown to have an effect on either the quality, size, or stability of the microsomal glutathione transferase crystals. The results from the detergent concentration study as well as the consequences of dialyzing for 5 days versus 6 days give some indications of the crystal formation and mode of crystallization. As suggested by Ku¨hlbrandt (1992), 2D crystals can form in different stages. In the current crystallization setup for microsomal glutathione transferase either a twostage or a three-stage model is feasible. A one-stage model can be excluded since big, noncrystalline membranes have been observed after 4 to 5 days of dialysis. This leaves the possibility of a two- or three-stage model. A two-stage model could be thought of as a situation where the cholate, which is used to solubilize the phospholipids, is removed very quickly and replaced by Triton, which has the lower CMC, and the Triton–phospholipid micelles merge with the Triton–protein micelles to form phospholipid bilayers containing disordered protein. In the second step, the protein rearranges itself in the membrane to form 2D crystals. In a three-stage model the protein is integrated into the preformed phospholipid bilayer followed by crystallization. Either of these two models seems likely. It is possible though that these preparations favor a two-stage model because of the low LPR and because crystallization takes place between the fifth and sixth days of dialysis and proteoliposomes were observed after 5 days. Thus, considering the need for a high detergent concentration and the transition from big, noncrystalline to crystalline membranes, crystallization and crystal size are rate dependent. Components of the Buffer Salt, pH, and protein concentration. A decrease in salt concentration does not alter the results of the dialysis. Increasing the salt concentration fivefold to 250 mM, however, yields, apart from the membrane sheets and vesicles, long, rolled-up sheets (Fig. 6) or vesicles several micrometers long and below 0.5 µm in diameter. Divalent salts could not be tested due to the use of cholate, which is precipitated by divalent cations. This detergent also determined the lowest pH to be tested to above pH 6, since cholate precipitates below this value as well. A pH of 6.5–10.2 did not have an effect on the crystallization (not shown).


FIG. 6. Apart from the standard crystalline sheets, rolled up sheets and long vesicles can be observed when the salt concentration is increased to above 250 mM. These crystals were not used for analysis due to their small diameter and the availability of larger membrane sheets. Scale bar 5 1 µm.

The protein concentration was varied in the range 0.5–1.75 mg/ml, which did not have any consequences on the outcome of the crystallization. Substrate. Two-dimensional crystals of microsomal glutathione transferase are grown in the presence of the substrate glutathione. However, it was found that the substrate does not induce crystallization since crystals form during dialysis without glutathione present as well (not shown). This is of importance in future structure–function experiments of the crystals since the substrate is thought to bind substoichiometrically (Sun and Morgenstern, 1997; Metters et al., 1994), and the binding of glutathione might not follow the crystal symmetry. Glycerol. The standard dialysis buffer contains 20% glycerol. Different glycerol concentrations in the range 0–40% were tested in combination with heating of the dialysate. Crystallization batches with concentrations of 40% yielded membranes identical to those prepared under standard conditions. Concentrations below 20% have an unfavorable effect on crystal sizes. Without glycerol and with a concentration of 10%, well-ordered crystals formed, but the membrane fragments were folded and aggregated, and their size was mostly limited to below 0.2 µm (Fig. 7). Heating in combination with glycerol did not improve the crystals further as described above. The Protein and Reproducibility Different purification batches form crystals very reliably. However, activity measurements showed that microsomal glutathione transferase was activated in unsuccessful crystallizations, while the crystals were not activated (unpublished observations). Activation could prevent crystal formation, which is possibly caused by a conformational change that occurs during activation.



FIG. 7. Lower concentrations of glycerol such as 10% and below have a negative effect on the crystal size as well as causing folding and precipitation. Scale bar 5 1 µm.

Dialysis Devices Various dialysis devices were tested in an effort to optimize the crystallization and its reproducibility. For initial trials, the dialysis tubing had a molecular weight cutoff of 12–14 kDa and a diameter of 6.4 mm. The tubing is simple to use and easily sampled after the dialysis. Small volumes of 50 µl were dialyzed in these tubes, and it is possible to dialyze even smaller volumes in tubes with a diameter of 2.5 mm. However, the 2.5-mm tubes are not as rigid which makes them somewhat inconvenient for the sampling. On successful crystallization, the volume was increased to 100–200 µl, which might be necessary for cryoelectron microscopy preparations. Air pockets can be removed from the tubes for the dialysis, but are kept for convenience during the sampling. These crystallizations are very reproducible. Another reproducible method uses commercially available dialysis cassettes. These devices are composed of two membranes separated by a frame, which gives a large dialysis surface. Various other commercial and noncommercial devices were tested such as dialysis buttons used for 3D crystallization, so-called ‘‘hockey sticks,’’ glass capillaries with a bent end to prevent clogging of the dialysis membrane by the precipitate (Ku¨hlbrandt, 1992), and a dialysis system (kindly provided by Professor Alain Brisson). These usually had a small membrane surface and might not be suitable for the low CMC of Triton X-100. Flow-through dialysis, frequent buffer exchanges, and various buffer volumes were tested as well, but these parameters did not affect the crystallization. CONCLUSIONS

This investigation of parameters was not only conducted in an effort to obtain crystals for the structural analysis of microsomal glutathione transferase, but was continued once the crystals were obtained. The most important parameters were identified, and it could be concluded that careful handling of the lipids ensures high reproducibility. If

problems should occur in the future, the system is well described, and certain parameters, such as salt concentration, can be excluded from a careful search to establish crystal formation again. The investigation of various parameters also led to a better understanding of the crystal formation of microsomal glutathione transferase as well as the rate dependence. In addition, it is hoped that a thorough investigation will contribute to the understanding of 2D crystallization in general, which might result in conclusions about universal tendencies once this method has become more established. In summary, it can be concluded that two wellordered crystal types of microsomal glutathione transferase form at the unusually low LPR of 3, which is the main parameter to induce crystallization. The crystal size can be positively manipulated by an increase in the detergent concentration, the optimal temperature, the glycerol concentration, and of changes in the dialysis device. Once the individual crystal types have formed, their quality is very stable, deterioration first occurs after several months of storage, and they are ordered to beyond 3 Å resolution. Salt concentration, pH, and the presence of substrate did not have an effect on either crystal quality or size. Not nearly all parameters and combinations of parameters have been investigated; for example, other detergents might be of importance and may explain some of the observed phenomena such as the significance of the rate of reconstitution. Bio-Beads would be an interesting method to answer questions about the rate dependence of crystallization as well, since Bio-Beads can be used to influence the rate of reconstitution. In addition, changes in membrane morphology (Rigaud et al., 1997) can be induced with Bio-Beads, which could be tested for this system. Recently it has been shown that both the orthorhombic and hexagonal forms of the 2D crystals of microsomal glutathione transferase (Hebert et al., 1997, Schmidt-Krey et al., submitted) are of sufficient size and quality for an atomic model, on which most of the effort is being concentrated at present. It is hoped that this will provide further information about the crystallization, such as crystal contacts and possibly ordered lipid molecules (Schmidt-Krey et al., submitted). In addition, crystallization attempts for the recombinant microsomal glutathione transferase have been successful (Schmidt-Krey, unpublished observations), which will allow studies of the structural variants to answer functional questions. We are grateful to Professor Werner Ku¨hlbrandt for discussions and to Professor Alain Brisson for discussions as well as a sample of his dialysis system. This project was supported by the Swedish

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