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Published: May 25, 2011 r 2011 American Chemical Society 3432 dx.doi.org/10.1021/cg2001442 | Cryst. Growth Des. 2011, 11, 3432–3441 ARTICLE pubs.acs.org/crystal Random Microseeding: A Theoretical and Practical Exploration of Seed Stability and Seeding Techniques for Successful Protein Crystallization Published as part of the Crystal Growth & Design virtual special issue on the 13th International Conference on the Crystallization of Biological Macromolecules (ICCBM13) Patrick D. Shaw Stewart,*,† Stefan A. Kolek,† Richard A. Briggs,†Naomi E. Chayen,‡ and Peter F. M. Baldock† †Douglas Instruments, Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, U.K. ‡Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, London SW7 2AZ, U.K. bS Supporting Information ’ INTRODUCTION Traditionally, microseeding has been used as an optimization step, where seed crystals are transferred into conditions that are similar to previously known crystallization conditions.1,2 Ireton and Stoddard3 introduced a novel, more systematic approach, referred to as microseed matrix-screening. This method was automated and further improved by D’Arcy et al.,4 who were the first to report the use of seeding with random screening kits. Experience has confirmed that “randomMMS” (rMMS) not only produces extra hits4,5 but also generates better-diffracting crystals.6 As shown in Figure 1, seeding in screening experiments picks up viable crystallization conditions in the metastable zone that without seeds would be overlooked as clear drops. The meta- stable zone may be much larger than the labile zone, so many additional hits may be found. Indeed, the labile zone may appear not to exist, although the metastable zone does; see the work of Ireton and Stoddard3 for an example. In spite of its effectiveness, several aspects of rMMS are poorly understood. We started this project with the objective of using seeding and other nucleation methods to generate better- diffracting crystals. We had eight specific questions in mind: (1) how canwe carry out rMMS in away thatwill give themaximum number of crystal hits? (2) How can we produce crystals with different space groups? (3) How can we reduce the number of salt crystals that arise in rMMS experiments? (4) How can we use microseeding for crystallizing protein complexes? (5) How can we stabilize seed stocks? (6) Is it helpful to “preseed” the protein stock? (7) Can we harvest seed crystals frommicrofluidic devices and capillaries? And, (8) can we encourage crystal nucleation if we have not yet obtained our first crystals? One of the innovations in D’Arcy’s 2007 paper4 was to suspend seed crystals in the solution that was in the reservoir of the well that the seed crystals were taken from, saving protein and simplifying the procedure. We will refer to this solution as the “Hit Solution”. We wanted to investigate the stability of the seed crystals in the Hit Solution and in other precipitants because the seed stock generally contains very little protein. In a typical experiment, the contents of a well with crystals (with a volume of say 0.6 μL) are suspended in 50 μL of Hit Solution. This means that the protein concentration in the seed stock is about 1.2% of the concentration that gave crystals in the original hit, say 0.06 mg/mL. This is likely to be near the point labeled “SHS” in Received: January 28, 2011 Revised: May 20, 2011 ABSTRACT: Microseed matrix-screening combined with ran- dom screens (rMMS) is a significant recent breakthrough in protein crystallization. In this study, a very reproducible assay for crystal seeds was set up that allowed the following recommenda- tions to be made: (1) the suitability of a solution for suspending seed crystals can be predicted by incubating (uncrushed) crystals in it for one day and observing crystal stability. (2) For routine rMMS, seed crystals should be suspended in the crystallization cocktail that gave the original crystals. (3) Seed crystals can be suspended in PEG or NaCl solutions to reduce the prevalence of salt crystals. (4) Protein complexes can be seeded with seed crystals suspended in PEG. If necessary, seed crystals can also be suspended in the original crystallization cocktail with any individual ingredients that destabilize the complex removed. (5) “Preseeding” of the protein stock should not be used if rMMS is available, because it is less effective. (6) Seed crystals can be harvested from microfluidic devices. (7) Heterogeneous nucleants and cross-seeding are less effective than rMMS, but they can be used if seed crystals cannot be obtained. A theoretical case and practical suggestions are also put forward for producing crystals with different space groups. 3433 dx.doi.org/10.1021/cg2001442 |Cryst. Growth Des. 2011, 11, 3432–3441 Crystal Growth & Design ARTICLE Figure 1, that is, well below the metastable zone; therefore, the crystals are likely to be thermodynamically unstable and to dissolve. To put it another way, if a protein molecule happens to fall off a microcrystal in the seed stock, it is unlikely that this molecule or any other protein molecule will subsequently attach itself to replace the loss. However, it is common experience that (macro-) crystals that have been harvested into the reservoir solution (containing no protein) may appear to be stable for limited periods. This implies that, as long as the precipitant concentration is relatively high, the rate of dissolution may be so low that it is not normally noticed. A second objective of the project was therefore to find out whether, in the conditions typically used, instability of the seed stock was an important consideration. This was of interest because some groups have reported that the rMMSmethod does not work in their hands, and we wondered if they were handling their seed stocks inappropri- ately. Moreover, other groups have reported that seed stocks that are left on the bench for a few hours may become inactive. Another area of concern was the tendency for rMMS experi- ments to generate salt crystals, which may waste investigators’ time and energy. This is a particular problem when the main precipitant in the Hit Solution is a salt. For example, if a seed stock containing ammonium sulfate is added to all conditions in a typical screen, one would expect to find several wells containing crystals of calcium sulfate (gypsum), because Ca2þ is common in crystallization screens. Moreover, Mg, Ca, Zn, and Cd frequently form phosphate crystals. We therefore investigated methods of reducing the number of salt crystals in rMMS experiments. Radaev and Sun8 showed that crystallization conditions for protein complexes heavily favor (71% versus 27%) polyethylene glycols rather than ammonium sulfate or other high-salt crystal- lization conditions. Seed stocks obtained from high-salt condi- tions may introduce harmful concentrations of salts that can interfere with the crystallization of protein�protein, protein�pep- tide, and protein�small molecule complexes and with the forma- tion of heave atom derivatives. A method that replaces high-salt precipitants with for example PEG would therefore be helpful. An obvious disadvantage of the rMMS method is that it can only be used when one has found at least one crystal, in order to make the first seed stock. We had several ideas for tackling this problem: first, we conjectured that seed crystals could be harvested from microfluidic devices. Second, we noted the approach of Habel & Hung,9 who collected precipitated protein from the wells of unsuccessful screening experiments and added it to random screens. It is likely that, even when crystals are not visible, some of the precipitates obtained are nevertheless crystal- line (the crystals may be smaller than the wavelength of light, therefore undetectable with optical microscopes). Third, we were interested in cross-seeding with crystals of unrelated proteins (see Results and Discussion). Fourth, we noted the investigations of Chayen & Saridakis10 of diverse nonprotein materials for protein nucleation. A nanoporous biocompatible glass (“bioglass”) was the most successful of these.11 This material has cavities that are comparable in size to protein molecules, and it has previously been shown to induce crystal- lization of at least 14 proteins, several of which could not be crystallized without bioglass.12 Sugahara et al.13 proposed the use of synthetic zeolite to induce nucleation. By including these het- erogeneous materials in our study, we planned to compare directly their effectiveness with regular microseeding. We emphasize that we were not seeking to prove here that rMMS microseeding is an effective way to crystallize proteins. This has already been clearly demonstrated by several publica- tions4�6 and a multitude of PBD entries (e.g., 3CL0, 3CKZ, 3CL2, 3I0M from the National Institute for Medical Research, UK). Instead, we investigated the relative effectiveness of different methods of inducing nucleation, with enough data to give good statistics, in order to improve nucleation techniques. ’EXPERIMENTAL SECTION Choice of Test Proteins. Table 1 shows the proteins used in this study. We selected proteins that could be obtained in large quantities and which did not typically crystallize in a large number of conditions in a screen. Other proteins, including lysozyme, concanavalin A, and ferritin, were not used because reliable Receptive Conditions could not be found (see below). Reagents and Screens Used. The commercial crystallization screens used were Molecular Dimensions’ Structure Screen 1 and JCSGþ, Jena Bioscience’s JBScreen Membrane 3, and Qiagen’s PEG Suite. In the experiments of Figures 4 and 8 (Supporting Information) and Table 4, we looked at the effect on crystallization of suspending seed crystals in the following solutions: Hit Sol.; 100% isopropanol (“IPA”); 100% polyethylene glycol, molecular weight 600 (“PEG 600”); 4 M (NH4)2SO4; a 50�50 mixture of Hit Sol. and 4 M (NH4)2SO4; 5.8 M NaCl; a 50�50mixture ofHit Sol. and 5.8MNaCl, and the stock solution of the protein being tested (see Table 1 for concentrations and buffers). Automation and Volumes Dispensed. We used an Oryx814 robot (Douglas Instruments) throughout the study.We dispensed vapor diffusion sitting-drop experiments to 96-well two- or three-drop plates (SwissCI). Drop volumes were 0.3 μL of protein plus 0.3 μL of screen for all nonseeding experiments, while we used 0.3 μL of protein, 0.29 μL of screen and 0.01 μL of seed stock for all seeding experiments except where we used 0.1 μL of seed stock (with 0.3 μL of protein, 0.2 μL of screen) as noted below. Optimization was carried out with the Oryx8 robot using “2-D Grid” designs (see Supporting Information). Figure 1. A screening experiment can be thought of as a set of points that land randomly on the phase diagram of a protein. (These “points” are shown as arrows because a vapor diffusion setup is assumed.) This schematic phase diagram7 has four main areas: an unsaturated zone where drops remain clear and no crystals can grow (labeled “under- saturated”); a zone where precipitation takes place (“precipitate”); a zone where crystal nuclei form and grow into visible crystals (“labile”); finally, there is a zone just below the labile zone, often called the “metastable” zone. Here, crystals do not form spontaneously, but if you take a crystal, for example from the labile zone, and put it into this zone, it will grow. The line that divides the metastable zone from the under- saturated zone is the solubility curve of the protein. In a normal screening experiment, crystals will only appear in wells where the arrows end up in or pass though the labile zone (thick arrows). If, however, you add seed crystals to the screen, you will obtain a set of additional hits where conditions end up in the metastable zone (dashed arrows). SHS and SPS indicate the rough positions of seed stocks made up in Hit Solution and protein stock, respectively (see text). 3434 dx.doi.org/10.1021/cg2001442 |Cryst. Growth Des. 2011, 11, 3432–3441 Crystal Growth & Design ARTICLE Identification of Receptive Conditions. We identified Recep- tive Conditions (see the definition belowTable 2) as follows: first, we set up three plates (two drops per well) with regular screens (i.e., no additive or seeds added) in order to eliminate conditions that crystallized at least once in the absence of seeds. We then set up a plate (again two drops per well) with the addition of crushed seed crystals suspended in IPA and another with seed crystals suspended in 100% PEG 600. We identified Receptive Conditions if (1) none of the six wells that were set up without the addition of seed crystals produced crystals, but (2) three out of the four wells with added seed crystals (suspended in IPA and PEG) produced crystals. Hemoglobin proved to be difficult to crystallize, and microseeding experiments were set up using the conventional method of suspending seed crystals in the Hit Solution, with nine wells per condition. We identified Receptive Conditions if none of the wells in the regular screens contained crystals, but all nine of the seeded wells did. Some proteins crystallized more quickly than others; for each protein, a cutoff period for observation of 1�7 days was used that gave reliable Receptive Conditions (see “Crystal Observation Period” of Table 1). The Receptive Conditions are shown in Table 2. Identification of Protein Crystals.We used a battery of tests to distinguish protein crystals from salt crystals during the experiments where Receptive Conditions were identified (see above). All crystals grown were well formed with straight edges, with the exception of hemoglobin, where crystals could be identified by color and clarity. We photographed putative crystals of the colorless proteins in a darkroom using UV light (wavelength 280 nm) with a microscope with normal glass optics and a Panasonic DMC-FX12 compact camera, using a 30 s “Starry Sky” setting. UV was provided by a UV Pen-280 (Douglas Instruments) with a 2 mm thick UG11 filter (Schott) to cut out visible light. Crystals that fluoresced with emission in the visible spectrum were identified as protein.15 When preparing seed stock, we crushed all large crystals with a glass probe, thereby subjecting them to the “crunch test”: crystals that produced a click that could be heard and felt were eliminated as salt crystals. The identification of protein crystals was confirmed with brightly colored (Jena Biosciences) and fluorescent16 dyes, and the cross-linking agent glutaraldehyde (Fluka). Glutaralde- hyde in high concentrations turns protein crystals brown or golden (see below). In the cases of crystals of the proteins concanavalin A, trypsin, and thaumatin, we used an interesting novel method of making the distinc- tion, which is a modification of the method of Pusey et al.17 We covalently labeled 50 μL aliquots of the proteins with the fluorescent dye DyLight 350 NHS Ester (from Thermo), following the manufac- turer’s instructions except that we used higher protein concentrations (30mg/mL for trypsin and concanavalin A, 36mg/mL for xylanase).We added 20 nL samples of labeled protein to wells containing putative protein crystals after the crystals had grown. We photographed crystals in a darkroom by illuminating with theUVPen-280 or with an FL4BLBUV lamp (Luxina), which has a peak wavelength of 370 nm. As shown in Figure 2, crystals fluoresced brightly and were unambiguously identified as protein rather than salt. (TheDyLight kits are very easy to use because all resins, columns, etc. are provided. We chose the label that is excited at 350 nm because it is not necessary to use a filter since most cameras have built-in UV filters.) The advantages of the method are (1) since it allows protein to be seen directly, it does not give false positives or negatives (except when the drop contains a lot of precipitate, see below). (2) It cannot interfere with the crystallization process. (3) Labeled protein need only be prepared if crystal identification by other methods fails; (4) even needles and small crystals can be identified. The method does not work well when the drop contains a lot of protein precipitate, which may absorb the labeled protein before it can reach the crystals. Note also that protein sometimes coats salt crystals in crystallization experi- ments, giving a superficially similar appearance. Such cases can, however, easily be distinguished by comparing UV images with visible light images because the protein coating is outside the salt crystal. Once Receptive Conditions were established, we identified protein crystals using the UV Pen-280 only. Preparation of Seed Stocks.We harvested all seed crystals from sitting drop plates except for xylanase, where we harvested the initial batch of seed crystals from microbatch-under-oil. All seed crystals (after the initial batch) were obtained from the conditions shown in bold and outlined in blue in Table 2 (i.e., crystals from microseeding experiments were used to prepare seed stocks for subsequent rounds ofmicroseeding). Seed stock suspensions were generated as follows: (1) a Seed Bead18 (Hampton Research) was placed in an Eppendorf tube on ice. (2) The well containing the crystals to be harvested was opened by cutting the tape, and 20 μL of reservoir solution from the well (or other solution) was placed in the tube. (3) Using a glass probe (with a 0.25 mm bead melted on the end) and observing with a microscope, the crystals were thoroughly crushed in the well. (4) 2 μL of reservoir solution (or other stabilizing solution) was transferred from the tube to the drop with seed crystals, crystals were suspended by withdrawing and dispensing from the pipet tip several times, and the mixture was transferred back to the tube using a slightly higher volume setting on the pipet to ensure all solution was transferred. (5) Step 4 was repeated to ensure that all crystals were picked up. (6) The tube and Seed Bead were vortexed for 2 min, the tube was placed back on ice, the bead was removed, and the stock was used immediately. Since the Oryx8 crystallization system uses contact dispensing (the tip touches the well during each dispensing operation), it was not necessary to centrifuge the seed stock to remove the larger seed crystals, which may cause blockages in noncontact systems. During experiments, the seed stock is held in the Oryx robot in one channel of the dispensing tip at ambient temperature. When not in use, all seed stocks were kept frozen at �15 �C. We also prepared a seed stock for cross-seeding with crystals of 15 proteins that were unrelated to any of the test proteins. These proteins (PDB codes) comprised N1 (3CL2) andN2 neuraminidase, a mutant of N1 (3CKZ), erythrocyte binding antigen, H3 hemagglutinin, Ser/Thr protein kinase, polycomb EED protein (3IJC), a post synaptic density protein (2RJI), the flavoprotein soxF, the cell envelope protein MtrF, rabbit hemorrhagic disease virus, and other mutations of these proteins. The seed stock was prepared as follows: (1) crystals of the first protein were crushed in their drops; (2) 3.5 μL of 100% PEG was added to each Table 1. Proteins Used in This Study protein supplier product code conc used (mg/mL) protein buffer “receptive” conditions found crystal observation period (days) glucose isomerase Hampton Research HR7-102 33 10 mM tris, 1 mM MgCl2 2 1 hemoglobin (bovine) Sigma Aldrich H2500 60 50 mM Na acetate 4 6 thaumatin Sigma Aldrich T7638 30 50 mM Na acetate 6 10 thermolysin Sigma Aldrich T7902 15 50 mM Na acetate, 14 mM NaOH 6 4 trypsin (porcine) Sigma Aldrich T7418 30 2% (w/v) benzamidine 2 3 xylanase Macro Crystal 36 43% glycerol, 0.2 M phosphate 5 7 3435 dx.doi.org/10.1021/cg2001442 |Cryst. Growth Des. 2011, 11, 3432–3441 Crystal Growth & Design ARTICLE drop, then a