- Email: [email protected]
Practical aspects of using the microbatch method in screening conditions for protein crystallization
Methods 34 (2004) 323–328 www.elsevier.com/locate/ymeth
Practical aspects of using the microbatch method in screening conditions for protein crystallization Allan D'Arcy,a,¤ Aengus Mac Sweeney,a and Alexander Habera,b a
Morphochem A.G. Basel, Switzerland b University of Freiburg, Germany Accepted 24 March 2004
Abstract The microbatch technique is a simple and eYcient method for screening for protein crystallization conditions both by hand and using automated systems. Many of the problems associated with more commonly used methods such as vapour diVusion can be overcome using this method. Despite its promise microbatch has not been widely utilized as a viable screening procedure. This review aims to describe the method from a practical point of view with an emphasis on screening crystallization conditions, outlining current progress and discussion of the advantages compared to other methods. 2004 Elsevier Inc. All rights reserved.
1. Introduction Protein crystallization has seen a great deal of renewed interest in the past few years. The many structural genomics and high-throughput crystallography initiatives have rekindled interest in what is often referred to as the “bottleneck” of a structure determination. In both the pharmaceutical industry and structural genomics laboratories, great eVorts have been directed toward Wnding better and more eYcient ways to identify and optimize conditions to crystallize proteins [1–4]. Crystallizing proteins under oil has many advantages compared to traditional hanging drop vapour diVusion methods and it can be well adapted to screening for initial crystallization conditions. The microbatch method was originally described by Chayen et al. [5] and is a technique where a small drop of protein sample, combined with the crystallization reagent of choice, is pipetted under a layer of oil. All of the reagents involved in the crystallization are present at a speciWc concentration ¤ Corresponding author. Present address: Protease Platform, Novartis Institutes for Biomedical Research, WKL-127.P74, 4002 Basel, Switzerland. Fax: +41-61-696-13-69. E-mail address: [email protected], allan.darcy@ morphochem.ch (A. D'Arcy).
1046-2023/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2004.03.023
and no signiWcant concentration of the protein or the reagents occurs in the drop. In the original work, a mineral oil of branched paraYns in the C20+ range was used which allows little or no diVusion of water. Under these conditions the technique is better suited for optimization of established conditions rather than a screening method. Chayen et al. have published numerous articles on the use of microbatch for optimization of crystallization conditions [6,7]. To use the microbatch method as a screening procedure, it is necessary to modify the experimental setup by using oils with diVerent properties. Choosing oils that allow a signiWcant diVusion of water will result in a concentration of the crystallization drop. The use of diVerent types of oils to inXuence the rate of diVusion and therefore the kinetics of the crystallization was Wrst described by D'Arcy et al. [8]. Using silicone Xuids, which are polymeric compounds composed of repeating dimethylsiloxane units (Si(CH3)2O)n it could be shown that it was possible to dramatically accelerate the rate at which crystals appeared compared to experiments using paraYn oil. With a 1:1 mixture of silicon Xuid and paraYn oil (Al's Oil, Hampton Research cat #HR3-413), it was demonstrated that a microbatch experiment under oil could be performed where a concentration of the drop occurred over a period of days to weeks. The method
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using the mixture of silicone Xuid is referred to as the “modiWed microbatch” or “microbatch diVusion.” This type of experiment will result in much higher protein concentrations as there is no end point in contrast to vapour diVusion experiments where an equilibrium is reached with the reservoir and concentration will stop. 2. Microbatch experiments In a microbatch experiment, the plate or individual wells are Wlled with the speciWc type of oil for a particular experiment. In the original experiments,described by Chayen et al. [5], shallow 72-well HLA plates (Nunc cat #470378) were used and 6 ml of the oil was dispensed onto the plate. Alternatively a standard 96-well plate can be used where each well is Wlled with 40–100 l of desired oil. The protein drop of typically 1 l is dispensed into the oil and will fall to the bottom of the well because of its higher density, the same volume of screening solution is then added to the protein drop. Mixing should occur spontaneously, (Fig. 1) if this is not the case, the coalescing of the drops can be facilitated by centrifuging the plate at 2500 rpm for 5 min. 2.1. Commonly used microbatch plates The plate originally used for microbatch experiments was a 72-well HLA plate also known as a Terizaki plate
(Nunc cat. #470378). This plate had a number of disadvantages, such as oil spilling out of the plates when moved, making it somewhat messy (perhaps a reason why microbatch has not been a popular method). The plates were not of a standard 96-well format and also tended to dry out relatively quickly. The 96-well vapour batch plate, introduced by Douglas Instruments, is a much improved design, this deeper plate prevents excessive oil spillage and has 96 wells instead of 72. Reservoir compartments have been incorporated which considerably retard the drops from drying out by slowing down the evaporation rate of water from the drops and the oil itself. In addition, it may be possible to inXuence the rate of evaporation by using reservoir solutions of much higher concentrations than the drop thus combining the advantages of microbatch and vapour diVusion. The most commonly used plates are shown in Fig. 2. 2.2. DiVerent experimental procedures When using modiWed microbatch for screening there are essentially three ways to set up the experiment: 1. Multichannel pipettes 2. Douglas Instruments crystallization robot. 3. Other liquid dispensing robots. The major diVerence between using the Douglas robot and the other methods is that with the Douglas system, the protein drop and precipitating agent are dispensed simultaneously (Fig. 3). In the other systems, the drops are dispensed sequentially, (Fig. 3) in general the protein drop Wrst, and then the precipitating agent as shown in Fig. 1. 2.2.1. Pipetting systems
Fig. 1. Pipetting sequence for microbatch experiment.
2.2.1.1. Multichannel pipettes. A number of manufacturers make manual or motor-riven multichannel pipetting devices and many are suitable for setting up microbatch screens manually. Although it may not be the most attractive proposition for a technician or student, it has
Fig. 2. 72 well HLA plate (A), 96 well round bottom plate (B), Douglas vapoou batchplate (C).
A. D'Arcy et al. / Methods 34 (2004) 323–328
Fig. 3. Simultaneous dispensing (Douglas system).
been demonstrated that as many as 40 screens or »4000 drops can be set up in an 8 h working day (R. Cudney) personal communication. This is well above the number of screens a normal (non-structural genomics) laboratory would need to run. However, setting up a standard 96 condition screen by hand in approximately 10 min is quite attractive considering the low cost of the equipment needed. 2.2.2. Robotic systems used for microbatch screening In principle any liquid handling systems capable of accurately dispensing microlitre volumes can be used for the microbatch method in a sequential manner. As described previously for the standard microbatch experiment, the plates or wells are Wrst Wlled with a layer of silicone/paraYn oil. Protein drops are then dispensed into the predetermined number of wells corresponding to the size of the screen used. The screening solutions may then be added individually or as a complete screen (e.g., 48– 96–384 conditions) depending on the type of robot used. One advantage of the microbatch system is that dispensing of both protein and screening solutions does not have to be done in a very rapid mode as the drops once under oil are stable and will not evaporate to any considerable extent during the pipetting process.
Unique to this system when it was introduced in 1992 was a special 2 or 5 bore microtip that allowed the simultaneous pipetting of protein and screening solutions using accurate motorized syringes. At the start of a screening experiment, the required amount of protein is aspirated into one of the bores of a two-channel microtip. The microtip then moves to a well containing the Wrst screening solution and aspirates 0.2–1 l of solution into the second bore of the microtip. The microtip now moves to the Wrst well of the (pre-oiled) target plate and simultaneously dispenses the predeWned volume of the protein and screening solution beneath the oil and the procedure is repeated for all the wells of the plate. We have occasionally observed more success using the simultaneous pipetting mode compared to the sequential approach and this may be an important aspect to be considered when designing an optimized screening system. 2.2.2.2. Robbins ScientiWc Tango LHS. The Robbins ScientiWc Tango pipetting robots (Fig. 5) are used in the high-throughput crystallization laboratory of Luft et al. [12,13] at the Hauptmann–Woodward Institute in BuValo New York. The laboratory uses mother–daughter plate technology to deliver crystallization cocktails and protein on 1536-well plates in microbatch crystallization experiments. The main components of the system are exchangeable banks of 1, 12, 96, or 384 syringes and a translation stage that holds up to 12 plates. The robots can simultaneously deliver up to 384 solutions with a volume range of 0.1–100 l, to minimize protein consumption a single channel dispenser is then used to pipette the protein solution. 2.2.2.3. Gilson constellation 1200. During evaluation of a new 96-well format microbatch plate by Greiner Bio-One (under development),we tested the Gilson constellation
2.2.2.1. Impax microbatch robot. The Impax liquid handling system, (Fig. 4) was developed by Douglas Instruments following initial research performed at Imperial College London [9–11].
Fig. 4. The impax system.
325
Fig. 5. Robbins ScientiWc Tango liquid handling system.
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2.4. Drop sizes
Fig. 6. Gilson constellation robot.
robot (Fig. 6), and found that, in contrast to some other non-contact pipetting systems, the drops containing protein and precipitating agent were well formed and crystals of glucose isomerase could be grown using standard conditions with this system.
Typically a screening drop would contain 1 l protein plus 1l screening reagent. This is a convenient drop size if protein is not in short supply and also makes it easier to remove and handle crystals for X-ray diVraction. Unfortunately protein supply, especially with diYcult molecules such as integral membrane proteins, can often be a major bottleneck when establishing crystallization conditions. Using the Impax system with the oil prepoured into the plate volumes of between 200 and 500 nl can be reproducibly pipetted. If the crystallization drop is dispensed Wrst and immediately covered with oil, total drop volumes of 100 nl or less are possible. In a recent article De Lucas et al. [3] have described using a modiWed microbatch screening method with a specially designed pipetting system that can make drops with total initial volumes of between 60 and 80 nl. Theoretically any of the robotic systems used to dispense nanolitre drops (below 50 nl) could be used for the microbatch system.
2.3. DiVerent oils used in crystallization The eVect of using paraYn oil (Merck 7174.2500/ Hampton Research HR3-411) or silicone Xuid (Dow Corning 200/1cs/Hampton Research HR3-415) and paraYn oil mixed 1:1 in a microbatch experiment has recently been reported [14]. In this report using, glucose isomerase, xylanase, trypsin, and lysozyme as test proteins the average number of conditions in a crystallization screen which gave crystals was increased twofold when using silicone oil compared to paraYn oil. Crystals which grow in the silicone oil experiments generally appear more quickly than in experiments with paraYn oil. The authors observe that this dramatic eVect is due not only to the increased diVusion of water in the case of silicone oil but also because of its high volatility. The silicone oil will evaporate and cause the drops to dry over a period of less than 1 week. If the crystallization experiment using silicone oil is placed in a humid environment (box containing reservoir solution or water) from the beginning, crystals appear more slowly but almost the same number of crystals was obtained over a period of approximately three weeks without the drops drying out. In many crystallization screens, up to half of the drops may remain clear throughout the crystallization experiment. Using Al's oil or silicone oil, much higher protein and precipitant concentrations may be reached than in a vapour diVusion experiment and this may facilitate spontaneous nucleation. Lorber and Geige [15] have used trimethylsiloxyl terminated polydimethylsiloxanes in Xoating drop experiments to simulate some of the eVects of growing crystals in microgravity and there are still many possibilities of exploring the properties of diVerent oils. Juvaniyama and co-workers have sucessfully used commercially available baby oil in thier microbatch experiments [24].
2.5. Comparison of modiWed microbatch and vapour diVusion Few studies can be found in the literature concerning the relative success rates of microbatch and vapour diVusion as screening methods. Chayen [16] conducted comparisons of standard microbatch using paraYn oil and vapour diVusion. More recently Baldock et al. [17] reported a comparative study using 6 commercially available proteins. In this study, the total number of hits in vapour diVusion was slightly higher than that for the paraYn/silicone (modiWed microbatch) experiment (41 versus 27) and there also appeared to be diVerences in the type of conditions that give crystals with the two methods. Nordeen and Cowen-Jacob [18] made a similar comparison with 8 pharmaceutically relevant proteins but in this study the authors found that both methods were equally successful in Wnding initial crystallization conditions. They also made the same observation that diVerent crystallization conditions were found depending on the method used. Sugahara has compared the two methods with 6 diVerent proteins in a commercial 48 condition screen. Crystals were found in 100 conditions with modiWed microbatch and 84 with vapour diVusion (personal communication). These results correlate well with our own observations comparing the two methods. We have found that in a protein-dependent manner one or the other method may give slightly better results but in a number of cases it was only possible to grow usable crystals in the microbatch system. A summary of some of these studies comparing the two methods can be found on the Douglas Instruments website http:// www.douglas.co.uk/mb&vdall.html. The general conclusion is that both methods are equally successful and may often produce diVerent hit
A. D'Arcy et al. / Methods 34 (2004) 323–328
conditions. There are, however, advantages to using a modiWed microbatch method for screening and all the methods that have been developed for dispensing nanodrops should be equally compatible with the microbatch system. In addition, the experimental setup is easier even with very small drops, as the drops are directly pipetted under oil there and the problem of excessive evaporation which can be a problem with vapour diVusion experiments is completely eliminated. In addition the step of dispensing reservoirs is no longer required thus reducing the number of manipulations and reducing the cost per experiment. 2.6. Temperature Temperature is known to be one of the most important yet often neglected parameters in protein cystallization and it is worth considering screening at a number of diVerent temperatures. Microbatch is well suited for running screens at diVerent temperatures and even over gradients of temperatures, e.g., from 4 to 37°C. In contrast to vapour diVusion experiments, there are no problems with condensation which can cause an increase in drop size and dilution of the sample. Examples of the possibilities of running temperature gradients in microbatch systems were Wrst reported by Luft et al. [19]. A microbatch technique coupled with a specially constructed thermal gradient was used to conduct polythermal experiments over the range 6–30 °C In our laboratory, we have used a simple programmable incubator to compare the crystallization behaviour of dihydro-neopterin aldolase, (a protein which gives many hits in a standard screen) at diVerent temperatures. Using a temperature gradient from 30 to 4 °C it was possible to obtain more hits, identify diVerent crystallization conditions and crystal morphologies [19]. One important practical point should be noted when doing this type of temperature experiment, the lid should not be removed from the plates if they are moved from 4 °C to room temperature for observation. This will cause condensation from the air to form on the oil layer and water will eventually sink to the drops causing undesired dilution, if the lid is left on there is no such eVect. 2.7. Crystals grown in microbatch, stabilizing, harvesting, and freezing Some of the reticence to using microbatch is because the removal and mounting of crystals appears to be more diYcult than in hanging or sitting drop experiments. Very often the initial hits found in microbatch screening are already suitable for X-ray analysis and structure determination and a few simple rules should be followed to be able to isolate and measure these crystals. Once crystals are observed it is important to monitor their growth on a daily basis, when the crystals appear to
327
have stopped growing or are of suYcient size for X-ray analysis the setups should be stabilized to prevent any deterioration of the crystals. This can be done in two ways, if the crystals have grown fairly rapidly (1–4 days) and are not needed for immediate analysis it is suYcient to replace the volatile Al's oil with the same quantity of paraYn oil, which should stabilize the crystals for at least 3–4 weeks. If the crystals need to be removed for Xray analysis this should be carried out with caution as Xooding the drops with an non-tested stabilizing solution may result in the crystals dissolving before they can be removed. It is diYcult to accurately determine the concentration of the solution in the drop at the time of harvesting and to make a “mother liquor” or cryoprotectant we have to make an educated guess as to what the composition of a suitable stabilizing solution should be. If the crystals have grown within 1–4 days one can assume that the concentration of the precipitating agent in the drop is lower or equivalent to the starting concentration of the reservoir solution. For example, if 25% peg 3350, 200 mM MgCl2 and 100 mM Tris 8.5 had originally been mixed with the protein drop it is reasonable to start with this solution to test stability. In order not to waste all the crystals in a drop by the wrong choice of mother liquor it is wise to remove one crystal at a time using a cryoloop. The crystal is then placed in approximately 20– 100 l the chosen mother liquor on a glass slide and observed under the microscope. If it appears to be stable the whole drop can be Xooded with the same solution and the remaining crystals stored in this way with the addition of paraYn oil as previously mentioned. If the investigator does not want to risk testing diVerent cryoprotectants or stabilizing solutions it is possible to remove the crystal with a rod mounted loop, dragging it slowly through the oil layer and directly into the nitrogen stream. In this way, the oil can act as a cryoprotectant and this is often the best way to get preliminary diVraction data. 2.8. Microbatch and membrane protein crystallization There are very few reports of microbatch crystallization of membrane proteins, one of the reasons was possibly the belief that the detergents were not compatible with the oils used. A recent article by Loll et al. [20] and Dijkstra and co-worker [21] has shown that this is not the case and microbatch systems can be used with detergents which are of course essential when screening for crystallization conditions with membrane proteins. A recent chapter in “Membrane Proteins” [22] by Naomi Chayen also gives some insights into the advantages and disadvantages of microbatch for membrane protein crystallization. Examples are given of membrane proteins that could be crystallized in the microbatch system and one case of the chlorophyll-binding protein 43 in which case
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better diVraction was obtained with microbatch grown crystals. If screens containing detergents are performed under oil, the integrity of the drop is much better preserved than in vapour diVusion especially when plates are moved during observation, which is another positive aspect for screening membrane proteins for crystallization conditions.
further improvement in the technology and a greater acceptance of the method.
Acknowledgments The authors would like to thank Bob Cudney for careful reading of the manuscript, helpful suggestions and encouragement.
3. Recent developments There have been major advances in the application of microXuidics for protein crystallization. The microchip method is a nanoscale-free interface diVusion that has a number of similarities to microbatch, as no reservoir solutions are used and a controlled, slow evaporation of the drops allows high protein concentrations to be sampled. The “Fluidigm Topaz” system is a commercially available product [23], which requires only 3 l protein to screen 48 diVerent conditions at 3 diVerent concentration ratios. Once conditions have been identiWed using the microprocessor, the microbatch method has been shown to be very suitable for scaling up and optimizing the initial crystals. (personal communication).
4. Conclusions ModiWed microbatch is the simplest method for running crystallization screens, the need for adding reservoir solutions is eliminated, greasing or other sealing methods are no longer necessary as the drops are immediately placed under a layer of oil. The fact that a layer of oil covers the protein may also provide protection from oxidation and other airborne contaminants. The method is compatible with the use of detergents and can therefore be used to screen crystallization conditions for membrane proteins. Performing crystallization trials at diVerent temperatures is greatly facilitated in microbatch systems as condensation problems observed using vapour diVusion are not encountered. Medium throughput can be achieved by hand using multichannel pipettes and there are relatively inexpensive robots on the market that have been designed speciWcally for the microbatch method. These systems are simple in their conception and allow screens to be performed in a suYciently highthroughput manner to suit the needs of most crystallization laboratories. Although statistically vapour diVusion and modiWed microbatch screening generate approximately the same number of hits, we often observed that certain proteins will only produce usable crystals in microbatch. It is unfortunate that the advantages of the method have not been fully recognized, but as more laboratories investigate the possibilities we can expect to see
References [1] J.R. Luft, J. WolXey, I. Jurisica, J. Glasgow, S. Fortier, G.T. DeTitta, J. Cryst. Growth 232 (2001) 591–595. [2] R.C. Stevens, Curr. Opin. Struct. Biol. 5 (2003) 558–563. [3] L.J. DeLucas, T.L. Bray, L. Nagy, D. McCombs, N. Chernov, D. Hamrick, L. Cosenza, A. Belgovskiy, B. Stoops, A. Chait, J. Struct. Biol. 142 (1) (2003) 188–206. [4] B. Rupp, Acc. Chem. Res. 36 (3) (2003) 173–181. [5] N.E. Chayen, P.D. Shaw Stewart, D.M. Blow, J. Cryst. Growth (1992) 176–180. [6] N.E. Chayen, Structure 5 (10) (1997) 1269–1274. [7] N.E. Chayen, J. Cryst. Growth (1999) 434–441. [8] A. D'Arcy, C. Elmore, M. Stihle, J.E. Johnston, J. Cryst. Growth 168 (1996) 175–180. [9] N.E. Chayen, P.D. Shaw Stewart, P. Baldock, New Dev. Acta Cryst. D 50 (1994) 456–458. [10] N.E. Chayen, P.D. Shaw Stewart, D.M. Blow, J. Cryst. Growth 122 (1992) 176–180. [11] N.E. Chayen, P.D. Shaw Stewart, D.L. Maeder, D.M. Blow, J. Appl. Crystallogr. 23 (1990) 297–302. [12] J.R. Luft, J. WolXey, R. Collins, M. Bianca, D. Weeks, I. Jurisica, P. Rogers, J. Glasgow, S. Fortier, G.T. DeTitta, in: Proceedings of NATO ASI 30th Course—Methods in Macromolecular Crystallography, “Ettore Majorana” Centre for ScientiWc Culture, Erice, Italy, 2000. [13] J.R. Luft, J. WolXey, I. Jurisica, J. Glasgow, S. Fortier, G.T. DeTitta, J. Cryst. Growth 232 (2001) 591–595. [14] A. D'Arcy, A. Mac Sweeney, M. Stihle, A. Haber, Acta Crystallogr. D 59 (2003) 396–399. [15] B. Lorber, R. Giege, J. Cryst. Growth 168 (1996) 204–215. [16] N.E. Chayen, Acta Cryst. D 54 (1998) 8–15. [17] P. Baldock, V. Mills, P.D. Shaw Stewart, J. Cryst. Growth 168 (1996) 170–174. [18] N. Noordeen, S. Cowan-Jacob, Recent Advances In Macromolecular Crystallization. Available from
Practical aspects of using the microbatch method in screening conditions for protein crystallization Allan D'Arcy,a,¤ Aengus Mac Sweeney,a and Alexander Habera,b a
Morphochem A.G. Basel, Switzerland b University of Freiburg, Germany Accepted 24 March 2004
Abstract The microbatch technique is a simple and eYcient method for screening for protein crystallization conditions both by hand and using automated systems. Many of the problems associated with more commonly used methods such as vapour diVusion can be overcome using this method. Despite its promise microbatch has not been widely utilized as a viable screening procedure. This review aims to describe the method from a practical point of view with an emphasis on screening crystallization conditions, outlining current progress and discussion of the advantages compared to other methods. 2004 Elsevier Inc. All rights reserved.
1. Introduction Protein crystallization has seen a great deal of renewed interest in the past few years. The many structural genomics and high-throughput crystallography initiatives have rekindled interest in what is often referred to as the “bottleneck” of a structure determination. In both the pharmaceutical industry and structural genomics laboratories, great eVorts have been directed toward Wnding better and more eYcient ways to identify and optimize conditions to crystallize proteins [1–4]. Crystallizing proteins under oil has many advantages compared to traditional hanging drop vapour diVusion methods and it can be well adapted to screening for initial crystallization conditions. The microbatch method was originally described by Chayen et al. [5] and is a technique where a small drop of protein sample, combined with the crystallization reagent of choice, is pipetted under a layer of oil. All of the reagents involved in the crystallization are present at a speciWc concentration ¤ Corresponding author. Present address: Protease Platform, Novartis Institutes for Biomedical Research, WKL-127.P74, 4002 Basel, Switzerland. Fax: +41-61-696-13-69. E-mail address: [email protected], allan.darcy@ morphochem.ch (A. D'Arcy).
1046-2023/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2004.03.023
and no signiWcant concentration of the protein or the reagents occurs in the drop. In the original work, a mineral oil of branched paraYns in the C20+ range was used which allows little or no diVusion of water. Under these conditions the technique is better suited for optimization of established conditions rather than a screening method. Chayen et al. have published numerous articles on the use of microbatch for optimization of crystallization conditions [6,7]. To use the microbatch method as a screening procedure, it is necessary to modify the experimental setup by using oils with diVerent properties. Choosing oils that allow a signiWcant diVusion of water will result in a concentration of the crystallization drop. The use of diVerent types of oils to inXuence the rate of diVusion and therefore the kinetics of the crystallization was Wrst described by D'Arcy et al. [8]. Using silicone Xuids, which are polymeric compounds composed of repeating dimethylsiloxane units (Si(CH3)2O)n it could be shown that it was possible to dramatically accelerate the rate at which crystals appeared compared to experiments using paraYn oil. With a 1:1 mixture of silicon Xuid and paraYn oil (Al's Oil, Hampton Research cat #HR3-413), it was demonstrated that a microbatch experiment under oil could be performed where a concentration of the drop occurred over a period of days to weeks. The method
324
A. D'Arcy et al. / Methods 34 (2004) 323–328
using the mixture of silicone Xuid is referred to as the “modiWed microbatch” or “microbatch diVusion.” This type of experiment will result in much higher protein concentrations as there is no end point in contrast to vapour diVusion experiments where an equilibrium is reached with the reservoir and concentration will stop. 2. Microbatch experiments In a microbatch experiment, the plate or individual wells are Wlled with the speciWc type of oil for a particular experiment. In the original experiments,described by Chayen et al. [5], shallow 72-well HLA plates (Nunc cat #470378) were used and 6 ml of the oil was dispensed onto the plate. Alternatively a standard 96-well plate can be used where each well is Wlled with 40–100 l of desired oil. The protein drop of typically 1 l is dispensed into the oil and will fall to the bottom of the well because of its higher density, the same volume of screening solution is then added to the protein drop. Mixing should occur spontaneously, (Fig. 1) if this is not the case, the coalescing of the drops can be facilitated by centrifuging the plate at 2500 rpm for 5 min. 2.1. Commonly used microbatch plates The plate originally used for microbatch experiments was a 72-well HLA plate also known as a Terizaki plate
(Nunc cat. #470378). This plate had a number of disadvantages, such as oil spilling out of the plates when moved, making it somewhat messy (perhaps a reason why microbatch has not been a popular method). The plates were not of a standard 96-well format and also tended to dry out relatively quickly. The 96-well vapour batch plate, introduced by Douglas Instruments, is a much improved design, this deeper plate prevents excessive oil spillage and has 96 wells instead of 72. Reservoir compartments have been incorporated which considerably retard the drops from drying out by slowing down the evaporation rate of water from the drops and the oil itself. In addition, it may be possible to inXuence the rate of evaporation by using reservoir solutions of much higher concentrations than the drop thus combining the advantages of microbatch and vapour diVusion. The most commonly used plates are shown in Fig. 2. 2.2. DiVerent experimental procedures When using modiWed microbatch for screening there are essentially three ways to set up the experiment: 1. Multichannel pipettes 2. Douglas Instruments crystallization robot. 3. Other liquid dispensing robots. The major diVerence between using the Douglas robot and the other methods is that with the Douglas system, the protein drop and precipitating agent are dispensed simultaneously (Fig. 3). In the other systems, the drops are dispensed sequentially, (Fig. 3) in general the protein drop Wrst, and then the precipitating agent as shown in Fig. 1. 2.2.1. Pipetting systems
Fig. 1. Pipetting sequence for microbatch experiment.
2.2.1.1. Multichannel pipettes. A number of manufacturers make manual or motor-riven multichannel pipetting devices and many are suitable for setting up microbatch screens manually. Although it may not be the most attractive proposition for a technician or student, it has
Fig. 2. 72 well HLA plate (A), 96 well round bottom plate (B), Douglas vapoou batchplate (C).
A. D'Arcy et al. / Methods 34 (2004) 323–328
Fig. 3. Simultaneous dispensing (Douglas system).
been demonstrated that as many as 40 screens or »4000 drops can be set up in an 8 h working day (R. Cudney) personal communication. This is well above the number of screens a normal (non-structural genomics) laboratory would need to run. However, setting up a standard 96 condition screen by hand in approximately 10 min is quite attractive considering the low cost of the equipment needed. 2.2.2. Robotic systems used for microbatch screening In principle any liquid handling systems capable of accurately dispensing microlitre volumes can be used for the microbatch method in a sequential manner. As described previously for the standard microbatch experiment, the plates or wells are Wrst Wlled with a layer of silicone/paraYn oil. Protein drops are then dispensed into the predetermined number of wells corresponding to the size of the screen used. The screening solutions may then be added individually or as a complete screen (e.g., 48– 96–384 conditions) depending on the type of robot used. One advantage of the microbatch system is that dispensing of both protein and screening solutions does not have to be done in a very rapid mode as the drops once under oil are stable and will not evaporate to any considerable extent during the pipetting process.
Unique to this system when it was introduced in 1992 was a special 2 or 5 bore microtip that allowed the simultaneous pipetting of protein and screening solutions using accurate motorized syringes. At the start of a screening experiment, the required amount of protein is aspirated into one of the bores of a two-channel microtip. The microtip then moves to a well containing the Wrst screening solution and aspirates 0.2–1 l of solution into the second bore of the microtip. The microtip now moves to the Wrst well of the (pre-oiled) target plate and simultaneously dispenses the predeWned volume of the protein and screening solution beneath the oil and the procedure is repeated for all the wells of the plate. We have occasionally observed more success using the simultaneous pipetting mode compared to the sequential approach and this may be an important aspect to be considered when designing an optimized screening system. 2.2.2.2. Robbins ScientiWc Tango LHS. The Robbins ScientiWc Tango pipetting robots (Fig. 5) are used in the high-throughput crystallization laboratory of Luft et al. [12,13] at the Hauptmann–Woodward Institute in BuValo New York. The laboratory uses mother–daughter plate technology to deliver crystallization cocktails and protein on 1536-well plates in microbatch crystallization experiments. The main components of the system are exchangeable banks of 1, 12, 96, or 384 syringes and a translation stage that holds up to 12 plates. The robots can simultaneously deliver up to 384 solutions with a volume range of 0.1–100 l, to minimize protein consumption a single channel dispenser is then used to pipette the protein solution. 2.2.2.3. Gilson constellation 1200. During evaluation of a new 96-well format microbatch plate by Greiner Bio-One (under development),we tested the Gilson constellation
2.2.2.1. Impax microbatch robot. The Impax liquid handling system, (Fig. 4) was developed by Douglas Instruments following initial research performed at Imperial College London [9–11].
Fig. 4. The impax system.
325
Fig. 5. Robbins ScientiWc Tango liquid handling system.
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2.4. Drop sizes
Fig. 6. Gilson constellation robot.
robot (Fig. 6), and found that, in contrast to some other non-contact pipetting systems, the drops containing protein and precipitating agent were well formed and crystals of glucose isomerase could be grown using standard conditions with this system.
Typically a screening drop would contain 1 l protein plus 1l screening reagent. This is a convenient drop size if protein is not in short supply and also makes it easier to remove and handle crystals for X-ray diVraction. Unfortunately protein supply, especially with diYcult molecules such as integral membrane proteins, can often be a major bottleneck when establishing crystallization conditions. Using the Impax system with the oil prepoured into the plate volumes of between 200 and 500 nl can be reproducibly pipetted. If the crystallization drop is dispensed Wrst and immediately covered with oil, total drop volumes of 100 nl or less are possible. In a recent article De Lucas et al. [3] have described using a modiWed microbatch screening method with a specially designed pipetting system that can make drops with total initial volumes of between 60 and 80 nl. Theoretically any of the robotic systems used to dispense nanolitre drops (below 50 nl) could be used for the microbatch system.
2.3. DiVerent oils used in crystallization The eVect of using paraYn oil (Merck 7174.2500/ Hampton Research HR3-411) or silicone Xuid (Dow Corning 200/1cs/Hampton Research HR3-415) and paraYn oil mixed 1:1 in a microbatch experiment has recently been reported [14]. In this report using, glucose isomerase, xylanase, trypsin, and lysozyme as test proteins the average number of conditions in a crystallization screen which gave crystals was increased twofold when using silicone oil compared to paraYn oil. Crystals which grow in the silicone oil experiments generally appear more quickly than in experiments with paraYn oil. The authors observe that this dramatic eVect is due not only to the increased diVusion of water in the case of silicone oil but also because of its high volatility. The silicone oil will evaporate and cause the drops to dry over a period of less than 1 week. If the crystallization experiment using silicone oil is placed in a humid environment (box containing reservoir solution or water) from the beginning, crystals appear more slowly but almost the same number of crystals was obtained over a period of approximately three weeks without the drops drying out. In many crystallization screens, up to half of the drops may remain clear throughout the crystallization experiment. Using Al's oil or silicone oil, much higher protein and precipitant concentrations may be reached than in a vapour diVusion experiment and this may facilitate spontaneous nucleation. Lorber and Geige [15] have used trimethylsiloxyl terminated polydimethylsiloxanes in Xoating drop experiments to simulate some of the eVects of growing crystals in microgravity and there are still many possibilities of exploring the properties of diVerent oils. Juvaniyama and co-workers have sucessfully used commercially available baby oil in thier microbatch experiments [24].
2.5. Comparison of modiWed microbatch and vapour diVusion Few studies can be found in the literature concerning the relative success rates of microbatch and vapour diVusion as screening methods. Chayen [16] conducted comparisons of standard microbatch using paraYn oil and vapour diVusion. More recently Baldock et al. [17] reported a comparative study using 6 commercially available proteins. In this study, the total number of hits in vapour diVusion was slightly higher than that for the paraYn/silicone (modiWed microbatch) experiment (41 versus 27) and there also appeared to be diVerences in the type of conditions that give crystals with the two methods. Nordeen and Cowen-Jacob [18] made a similar comparison with 8 pharmaceutically relevant proteins but in this study the authors found that both methods were equally successful in Wnding initial crystallization conditions. They also made the same observation that diVerent crystallization conditions were found depending on the method used. Sugahara has compared the two methods with 6 diVerent proteins in a commercial 48 condition screen. Crystals were found in 100 conditions with modiWed microbatch and 84 with vapour diVusion (personal communication). These results correlate well with our own observations comparing the two methods. We have found that in a protein-dependent manner one or the other method may give slightly better results but in a number of cases it was only possible to grow usable crystals in the microbatch system. A summary of some of these studies comparing the two methods can be found on the Douglas Instruments website http:// www.douglas.co.uk/mb&vdall.html. The general conclusion is that both methods are equally successful and may often produce diVerent hit
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conditions. There are, however, advantages to using a modiWed microbatch method for screening and all the methods that have been developed for dispensing nanodrops should be equally compatible with the microbatch system. In addition, the experimental setup is easier even with very small drops, as the drops are directly pipetted under oil there and the problem of excessive evaporation which can be a problem with vapour diVusion experiments is completely eliminated. In addition the step of dispensing reservoirs is no longer required thus reducing the number of manipulations and reducing the cost per experiment. 2.6. Temperature Temperature is known to be one of the most important yet often neglected parameters in protein cystallization and it is worth considering screening at a number of diVerent temperatures. Microbatch is well suited for running screens at diVerent temperatures and even over gradients of temperatures, e.g., from 4 to 37°C. In contrast to vapour diVusion experiments, there are no problems with condensation which can cause an increase in drop size and dilution of the sample. Examples of the possibilities of running temperature gradients in microbatch systems were Wrst reported by Luft et al. [19]. A microbatch technique coupled with a specially constructed thermal gradient was used to conduct polythermal experiments over the range 6–30 °C In our laboratory, we have used a simple programmable incubator to compare the crystallization behaviour of dihydro-neopterin aldolase, (a protein which gives many hits in a standard screen) at diVerent temperatures. Using a temperature gradient from 30 to 4 °C it was possible to obtain more hits, identify diVerent crystallization conditions and crystal morphologies [19]. One important practical point should be noted when doing this type of temperature experiment, the lid should not be removed from the plates if they are moved from 4 °C to room temperature for observation. This will cause condensation from the air to form on the oil layer and water will eventually sink to the drops causing undesired dilution, if the lid is left on there is no such eVect. 2.7. Crystals grown in microbatch, stabilizing, harvesting, and freezing Some of the reticence to using microbatch is because the removal and mounting of crystals appears to be more diYcult than in hanging or sitting drop experiments. Very often the initial hits found in microbatch screening are already suitable for X-ray analysis and structure determination and a few simple rules should be followed to be able to isolate and measure these crystals. Once crystals are observed it is important to monitor their growth on a daily basis, when the crystals appear to
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have stopped growing or are of suYcient size for X-ray analysis the setups should be stabilized to prevent any deterioration of the crystals. This can be done in two ways, if the crystals have grown fairly rapidly (1–4 days) and are not needed for immediate analysis it is suYcient to replace the volatile Al's oil with the same quantity of paraYn oil, which should stabilize the crystals for at least 3–4 weeks. If the crystals need to be removed for Xray analysis this should be carried out with caution as Xooding the drops with an non-tested stabilizing solution may result in the crystals dissolving before they can be removed. It is diYcult to accurately determine the concentration of the solution in the drop at the time of harvesting and to make a “mother liquor” or cryoprotectant we have to make an educated guess as to what the composition of a suitable stabilizing solution should be. If the crystals have grown within 1–4 days one can assume that the concentration of the precipitating agent in the drop is lower or equivalent to the starting concentration of the reservoir solution. For example, if 25% peg 3350, 200 mM MgCl2 and 100 mM Tris 8.5 had originally been mixed with the protein drop it is reasonable to start with this solution to test stability. In order not to waste all the crystals in a drop by the wrong choice of mother liquor it is wise to remove one crystal at a time using a cryoloop. The crystal is then placed in approximately 20– 100 l the chosen mother liquor on a glass slide and observed under the microscope. If it appears to be stable the whole drop can be Xooded with the same solution and the remaining crystals stored in this way with the addition of paraYn oil as previously mentioned. If the investigator does not want to risk testing diVerent cryoprotectants or stabilizing solutions it is possible to remove the crystal with a rod mounted loop, dragging it slowly through the oil layer and directly into the nitrogen stream. In this way, the oil can act as a cryoprotectant and this is often the best way to get preliminary diVraction data. 2.8. Microbatch and membrane protein crystallization There are very few reports of microbatch crystallization of membrane proteins, one of the reasons was possibly the belief that the detergents were not compatible with the oils used. A recent article by Loll et al. [20] and Dijkstra and co-worker [21] has shown that this is not the case and microbatch systems can be used with detergents which are of course essential when screening for crystallization conditions with membrane proteins. A recent chapter in “Membrane Proteins” [22] by Naomi Chayen also gives some insights into the advantages and disadvantages of microbatch for membrane protein crystallization. Examples are given of membrane proteins that could be crystallized in the microbatch system and one case of the chlorophyll-binding protein 43 in which case
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better diVraction was obtained with microbatch grown crystals. If screens containing detergents are performed under oil, the integrity of the drop is much better preserved than in vapour diVusion especially when plates are moved during observation, which is another positive aspect for screening membrane proteins for crystallization conditions.
further improvement in the technology and a greater acceptance of the method.
Acknowledgments The authors would like to thank Bob Cudney for careful reading of the manuscript, helpful suggestions and encouragement.
3. Recent developments There have been major advances in the application of microXuidics for protein crystallization. The microchip method is a nanoscale-free interface diVusion that has a number of similarities to microbatch, as no reservoir solutions are used and a controlled, slow evaporation of the drops allows high protein concentrations to be sampled. The “Fluidigm Topaz” system is a commercially available product [23], which requires only 3 l protein to screen 48 diVerent conditions at 3 diVerent concentration ratios. Once conditions have been identiWed using the microprocessor, the microbatch method has been shown to be very suitable for scaling up and optimizing the initial crystals. (personal communication).
4. Conclusions ModiWed microbatch is the simplest method for running crystallization screens, the need for adding reservoir solutions is eliminated, greasing or other sealing methods are no longer necessary as the drops are immediately placed under a layer of oil. The fact that a layer of oil covers the protein may also provide protection from oxidation and other airborne contaminants. The method is compatible with the use of detergents and can therefore be used to screen crystallization conditions for membrane proteins. Performing crystallization trials at diVerent temperatures is greatly facilitated in microbatch systems as condensation problems observed using vapour diVusion are not encountered. Medium throughput can be achieved by hand using multichannel pipettes and there are relatively inexpensive robots on the market that have been designed speciWcally for the microbatch method. These systems are simple in their conception and allow screens to be performed in a suYciently highthroughput manner to suit the needs of most crystallization laboratories. Although statistically vapour diVusion and modiWed microbatch screening generate approximately the same number of hits, we often observed that certain proteins will only produce usable crystals in microbatch. It is unfortunate that the advantages of the method have not been fully recognized, but as more laboratories investigate the possibilities we can expect to see
References [1] J.R. Luft, J. WolXey, I. Jurisica, J. Glasgow, S. Fortier, G.T. DeTitta, J. Cryst. Growth 232 (2001) 591–595. [2] R.C. Stevens, Curr. Opin. Struct. Biol. 5 (2003) 558–563. [3] L.J. DeLucas, T.L. Bray, L. Nagy, D. McCombs, N. Chernov, D. Hamrick, L. Cosenza, A. Belgovskiy, B. Stoops, A. Chait, J. Struct. Biol. 142 (1) (2003) 188–206. [4] B. Rupp, Acc. Chem. Res. 36 (3) (2003) 173–181. [5] N.E. Chayen, P.D. Shaw Stewart, D.M. Blow, J. Cryst. Growth (1992) 176–180. [6] N.E. Chayen, Structure 5 (10) (1997) 1269–1274. [7] N.E. Chayen, J. Cryst. Growth (1999) 434–441. [8] A. D'Arcy, C. Elmore, M. Stihle, J.E. Johnston, J. Cryst. Growth 168 (1996) 175–180. [9] N.E. Chayen, P.D. Shaw Stewart, P. Baldock, New Dev. Acta Cryst. D 50 (1994) 456–458. [10] N.E. Chayen, P.D. Shaw Stewart, D.M. Blow, J. Cryst. Growth 122 (1992) 176–180. [11] N.E. Chayen, P.D. Shaw Stewart, D.L. Maeder, D.M. Blow, J. Appl. Crystallogr. 23 (1990) 297–302. [12] J.R. Luft, J. WolXey, R. Collins, M. Bianca, D. Weeks, I. Jurisica, P. Rogers, J. Glasgow, S. Fortier, G.T. DeTitta, in: Proceedings of NATO ASI 30th Course—Methods in Macromolecular Crystallography, “Ettore Majorana” Centre for ScientiWc Culture, Erice, Italy, 2000. [13] J.R. Luft, J. WolXey, I. Jurisica, J. Glasgow, S. Fortier, G.T. DeTitta, J. Cryst. Growth 232 (2001) 591–595. [14] A. D'Arcy, A. Mac Sweeney, M. Stihle, A. Haber, Acta Crystallogr. D 59 (2003) 396–399. [15] B. Lorber, R. Giege, J. Cryst. Growth 168 (1996) 204–215. [16] N.E. Chayen, Acta Cryst. D 54 (1998) 8–15. [17] P. Baldock, V. Mills, P.D. Shaw Stewart, J. Cryst. Growth 168 (1996) 170–174. [18] N. Noordeen, S. Cowan-Jacob, Recent Advances In Macromolecular Crystallization. Available from