Strategies for Protein Cryocrystallography - Crystal Growth & Design

Article pubs.acs.org/crystal

Strategies for Protein Cryocrystallography Laura Vera and Enrico A. Stura* CEA, iBiTec-S, Service d’Ingénierie Moléculaire des Protéines, Laboratoire de Toxinologie Moléculaire et Biotechnologies, Gif-sur-Yvette, F-91191, France ABSTRACT: Cryoprotection is the final step before flash-cooling, during which crystals can be improved or damaged and data quality maximized. A well thoughtout cryoprotection requires optimized composition of suitable components and an appropriate soak period. Crystallization methodology has been extensively studied, but not cryoprotection. Cryoprotectant selection remains a trial and error exercise where the first combination that “works” is accepted. The approach presented here consists in a formulation of a few mixed compounds to speed-up crystal preparation for synchrotron data collection. A good cryoprotectant solution needs to stabilize the crystalline state and prevent ice formation during flash-cooling, its composition can differ widely from the crystallization precipitant since it is not required to induce nucleation, establish an equilibrium between the protein crystal and the solution or permit crystal growth. The strategy presented here differs from the general practice that involves a single anti-icing chemical, often glycerol, a molecule able to enhance protein solubility. The multicomponent mixtures selected through an evolutive process neither increase nor decrease protein solubility but provide extended stabilization during cryoprotection to allow longer soak periods without crystal cracking or dissolving.

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INTRODUCTION Macromolecular crystals are highly susceptible to rapid deterioration because of radiation damage if X-ray data collection is performed at room temperature. At synchrotron X-ray sources this is even more dramatic, so data are now routinely collected from samples kept at around 100 K to reduce secondary radiation damage. Preservation of crystal integrity for longer periods in the X-ray beam, allows for higher resolution and better data quality to be collected from each sample. Unlike inorganic crystals, the high water content in the mother liquor that surrounds protein crystals and within the crystal lattice itself can be problematic when the crystals are cooled to cryogenic temperatures. A rapid transition is important in order to achieve water vitrification rather than ice formation as the samples are flash cooled. Ice formation can result in unsightly dark rings in the diffraction pattern, with consequent loss of a substantial shell of data. Thus, the crystals are soaked in a cryoprotectant solution, so that the mother liquor that surrounds them is exchanged for a solution suitable for water vitrification when the crystals are subsequently flash cooled to cryogenic temperatures. The relatively high water content of protein crystals has been considered to be less of a problem that the liquid surrounding them and in a relatively large number of cases, data has been collected from crystals transferred into dry paraffin oil, silicone, paratone, and other non water-miscible oils. If water within the crystal lattice is believed to be a problem, the use of oils can be combined with a prior soak in a cryoprotectant.1 After the soak in the cryoprotectant solution, crystals may not need any surrounding liquid. At times, better data can be collected from the portion of a long crystal that sticks out of a cryo-loop © XXXX American Chemical Society

without any surrounding solution because of the absence of scattering from the encompassing amorphous medium. To avoid damage to crystals during the transfer from the mother liquor to the cryoprotectant solution, crystals can be grown directly in solutions that provide cryoprotection. Small molecular weight polyethylene glycol (PEG), certain lithium2 and various carboxylate salts3 have good cryoprotecting properties and are compatible with protein crystallization. This approach limits the range of options for crystal optimization and may constrain efforts to make crystals larger and better ordered, but it can be effective as part of a broadrange crystallization strategy. The typical procedure for preparing a specimen for diffraction in a cryogenic environment, consists in selecting an appropriate crystal in the crystallization tray, picking it with a cryo-loop and transferring it as rapidly as possible to a cryoprotectant solution from which is then scooped up again and plunged in liquid nitrogen, or, if a faster cooling rate is desired another cryogenic liquid4 or a cold gas stream.5 Historically, after the determination of suitable cryoprotection for 50 typical protein crystallization solutions using glycerol,6 this compound has been incorporated in many formulations. Because in some cases the elevated glycerol concentration needed for cryoprotection are not easily tolerated by crystals, even for brief periods, it has become important to find the minimum concentration that avoids ice Special Issue: ICCBM14 Received: October 18, 2012 Revised: January 25, 2013

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dx.doi.org/10.1021/cg301531f | Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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formation. To improve on this trial and error procedure, in which many crystals are lost, predictive algorithms based on heat transfer analysis and thermal properties of cryoprotectant solutions have been measured and modeled for aqueous glycerol, ethylene glycol and ternary glycerol−salt−water mixtures.7 These glycols (glycerol, ethylene, and propylene glycols) are well-known as mild protein solubility enhancers and thus effective in reducing crystal nucleation and growth,8 so it is not surprising to find that crystals tend to melt when placed in a poorly formulated cryoprotectant solutions without a protein-saturated mother-liquor able to maintain the equilibrium between the protein in solution and the crystal lattice. In our studies on various proteins, to counteract the effect of the glycols, we have augmented the precipitant concentration and introduced “cryo-precipitants”. Cryo-precipitants are compounds useful as precipitants in protein crystallization which can disrupt ice formation. Among such compounds, 2methyl-2,4-pentanediol (MPD), is an effective precipitant for protein crystallization as well as a useful cryoprotectant.9 Dimethyl sulfoxide (DMSO), a polar solvent that helps dissolve both polar and nonpolar compounds in water and that can be used as a precipitant or additive in protein crystallization,10 is also an effective cryoprotectant. Other well-known compounds with these dual properties are the short PEGs such as PEG 200−600 and monomethyl polyethylene glycol (MPEG) 550. The cryo-precipitants counteract the effect of the “cryosolubilizers” (glycerol, ethylene and propylene glycols) to achieve a balance that maintains crystal integrity. To select mixtures with the desired properties we have designed a selection procedure, here referred to as “champion challenge” method. By comparing our best formulations with improved ones, we have progressed from mixtures of cryoprotectant compounds that work for a single project to solutions that can be used in the rational optimization of cryoprotocols for a vast array of proteins.

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Figure 1. Graphical representation of the final formulation of cryoprotectant solutions. 40% by volume consist in a mixture of seven compounds that inhibit ice formation, defined the core components. The core components are: 50% v/v diethylene glycol (D), ethylene glycol (E), glycerol (G), 2-methyl-2,4-pentanediol (M), propylene glycol or 1,2 propane diol, (P), dimethyl sulfoxide (S) with the additive 3-(1-pyridinio)-1- propane sulfonate at 100 mM (F). 50% by volume is reserved to the precipitant-water mixture, typically 2× compared to the crystallization precipitant. 10% by volume is assigned to the buffer. Linear buffers have been chosen for the rapid preparation of buffered cryoprotectant solutions.14 The buffer is chosen within two pH units from that used in the crystallization, or more in the direction of lower protein solubility. connected to a syringe, crystals are picked up from the sitting drop tray in which they are grown and transferred together with a small amount of mother liquor into the cryoprotectant solution. The ratio of the volume of the transferred mother liquor to cryoprotectant solution will typically be 1:5. If larger cryoprotectant volumes are used, it can be increased to 1:50. Because the crystal is transferred with its surrounding mother liquor, the shock suffered during the transfer from one solution to the other is reduced. The procedure is slower that the loop transfer method but we have had very good success. It has the advantage that it can preserve, even if at lower concentrations, important additives that would be complicated to add to the cryoprotectant solution. Selection of Cryo-Solutions. To compare the performance of different formulations with crystals grown under a variety of crystallization conditions, the screening system has been made modular. The three modules are: precipitant mix, cryoprotectant mix and buffer mix. Linear buffers are used to facilitate mixing without measuring the pH.14 The modularity of the system is constrained for crystals requiring high precipitant concentrations for their growth, for which a 2× precipitant solution is not possible. While crystals grown at low salt in PEG can be tested using the standardized cryoprotectant formulation described above, alternative strategies may have to be adopted in other cases (Figure 2). Fortunately, crystals grown at high PEG or high salt concentrations often require no cryoprotectant. Cryosalts have proved effective for high salt crystallizations. They are tested unbuffered and if necessary tested with a buffer at various pH. Cryoprotectant solutions obtained for these more challenging cases are recorded in our database adding to the “prior knowledge” alternative approach (Figure 2). Flash Cooling. The crystal is retrieved from the cryoprotectant solution with a cryo-loop and plunged in liquid nitrogen in ESRF pucks, which are then stored in a Dewar of liquid nitrogen. Transfers from crystallization tray to cryoprotectant solution and into the liquid nitrogen are done rapidly. The behavior of crystals during these transfer steps is rather unpredictable and a source of variability. A slow

EXPERIMENTAL PROCEDURES

Crystallization Experiments. Crystallization experiments were carried out in CrysChem sitting drop vapor diffusion plates with protein drops of 1 and 1 μL precipitant solution which are stored in a cooled incubator at 20 °C. Typically screening was carried out in a systematic manner11 and crystals were systematically optimized by streak seeding.12 Protocol. A 100 μL cryoprotectant solution is mixed from 40 μL of cryoprotectant mixture (cryomix), 10 μL of buffer, and 50 μL of precipitant. Cryomixes are formulated from various core components: glycerol, ethylene glycol, diethylene glycol, propanediol, MPD, DMSO and the additive 3-(1-pyridinio)-1-propane sulfonate and prepared at 2.5X, buffers at 10X and precipitants at 2X (Figure 1). The crystal soaking transfer is carried out in cryo-trays consisting of CrysChem sitting drop plates or XRL plates with microbridges. The mixed cryoprotectant is added to the cavity on the bridge or pedestal, typically 10 μL. Water is added around the pedestal in the CrysChem plates or below the microbridges to provide moisture during all phases of crystal transfer and soaking. This environment allows for easy manipulation. Under a microscope, a cryo-loop is used to pick up the crystal from the crystallization tray, the crystallization tray is then swapped for the cryo-tray and the crystal is deposited in the cryoprotectant solution. For short durations, up to 15 min, the cryoprotectant solution with the crystal inside is left exposed to the ambient air. For longer soaks, needed to allow for the exchange of ligands, the enclosure is covered with a coverglass fixed with a dot of grease to prevent it from moving, but it is not sealed to allow for easy removal. Capillary Transfer Method. The method used here is a variation of protocol 2 described in Stura and Gleichman, 1999.13 Using a capillary B



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Figure 2. Flowchart describing the method by which cryoprotectant solutions are selected. The nine cryomixes C1−C9 formulated at 2.5× are tested in any order for any new crystal form that has never been flash cooled before except in those cases where crystals have been grown from high concentration precipitants for which a 2× precipitant solution cannot be mixed. Most crystals can be successfully prepared by the standard procedure (orange highlighted box) using one of the nine cryomixes but exceptionally a more complex screening procedure may be needed with the concomitant evolution of a cryomix, the arrival of a “challenger” and the eventual establishment of a new “champion”. Once the first crystal has been flashfrozen, further crystals are prepared using the “prior knowledge” strategy. Cryoprotecting compounds, such as glycerol or ethylene glycol are replaced by cryomixes, one cryomix is replaced by another, salt concentrations, the molecular weight of the PEG precipitant and pH are varied, and for crystals grown under crystallization conditions for which a suitable condition was recorded in the database, the same cryosolutions are tested. Variation of core-components contributes to cryomix evolution. transfer from crystallization tray to cryoprotectant solution could dehydrate the crystal making it unsuitable for further experiments. After the soak in cryoprotectant, crystals appear to be more resistant to dehydration. A slower transfer from cryoprotectant to liquid nitrogen could lead to crystal mild dehydration and have a positive influence on diffraction-quality. The use of liquid ethane instead of liquid nitrogen can ensure faster vitrification.4 Screening Strategy. The screening is subdivided in to three phases, in the first phase, a suitable cryosolution is selected for crystals that have never been flash-cooled before (Figure 2). In the second phase, a small number of alternative cryoprotectant solutions are mixed making use of the knowledge acquired in phase 1. Third phase screening strategy is an extension of phase 2 taking into account X-ray diffraction data quality. Phases 2 and 3 are important to reduce the number of cryomix formulations whose combinations could become too numerous to test. The selection procedure we have adopted has been based on what we call the “champion competition” method. The most successful cryoprotectant mix in the previous data collection run is retained for the following data collection session and competed against new precipitant−cryoprotectant−buffer combinations. The champion is over-represented compared to each of the challengers, but this bias is valuable since it adds a statistical weight to account for previous successes achieved with that cryoprotectant solution. Champions selected for a particular protein crystal are also allowed to compete to become champions for other protein crystals independently of the crystallization conditions from which the crystals have been grown. This part of the selection procedure is designed to allow the evolution of cryoprotectant solutions that provide stability

for most protein crystals independently of how they have been crystallized. Since sometimes ligand soaking is carried out in the cryoprotectant solution, a formulation that gives long-term crystal stability is more likely to be retained to become a champion. Screening Examples. The method and evolution of the cryomixes can be better explained with three “typical” examples (Table 1) that illustrate how the methodology described in Figure 2 is applied in practice. A simple illustration of an initial search is given by the antiphenanthroline dicarboxylate Fab UO7, where cryomixes C6 and C3 compete against each other with two crystals each resulting in C3 becoming a clear winner (Table 1). C6 is present in two formulations U7cryo1 and U7cryo5 giving anisotropic diffraction or no diffraction at all, while both formulations with cryomix C3 diffract to 1.7 Å. The screening also included two tests with a successful database cryoformulation, M9cryo7, unrelated to the crystallization conditions for Fab UO7. Both crystals performed reasonably well, diffracting to 2.0 and 2.5 Å. A challenging cryopreservation problem is best illustrated by crystals of human full-length MMP-13 that grew spontaneously in an eppendorf tube.15 The absence of a precipitant providing crystal stabilization and contributing the anti-icing action of cryoprotectant compounds, resulted in only one crystal out of fifteen giving any usable diffraction. The results was disappointing given that the cryopreservation of crystals grown in eppendorf tubes had been solved in a prior study for crystals of house dust mite major allergen Der p 116 and that the same methodology had been applied. The only diffracting crystal, was cryopreserved by M13cryo2, a condition used in duplicate with a negative result (Table 1). In a successive synchrotron data collection C



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Table 1. Example of Cryo-Formulation Strategies example Fab UO7

crystallization 30% MPEG 550, 100 mM imidazole HCl, pH 7.0

*database entry M9Cryo7 example human MMP13

crystal

resolution

1

no diffraction

U7Cryo5: C6,a 12.5% w/v MPEG 5K, 25% MPEG 550, 100 mM PCTP 50/50 U7Cryo2: C3,a 15% w/v MPEG 5K, 100 mM PCTPb 50/50 U7Cryo4: 50% C3,a 30% w/v MPEG 5K, 100 mM IMb 50/50 M9Cryo7* M9Cryo7*

2

anisotropic

3 4 5 6

1.7 Å 1.7 Å 2Å 2.5 Å

composition

strategy

5% v/v diethylene, glycol, 20% v/v 1.2-propanediol, 5% w/v glycerol, 10% w/v PEG 10K, 200 mM NaCl, 100 mM PCTPb 80/20 crystallization cryosolution Crystals grown spontaneously in eppendorf tube

*database entry M9Cryo5

cryosolution U7Cryo1: C6,a 25% w/v MPEG 5K, 100 mM PCTPb 50/50

M13Cryo2: 10% v/v ethylene glycol, 20% w/v MPEG2K, 10% v/v MPEG550, 90 mM imidazole malate, pH 6.0 M13Cryo2 M9Cryo5* M9Cryo7*

composition a

example υ-Da2a

36% MPEG 2K, 450 mM NaCl, 90 mM NaSCN, 100 mM imidazole HCl, pH 7−7.5

MT1Cryo1 LCACryo7 LCACryo9

no diffraction

2 3 4

2.85 Å 2.44 Å 2.6 Å

this cryosolution with C1similar to M13cryo2 was successful in cryoprotecting other proteins. cryosolution crystal resolution crystallization condition crystallization condition MT1Cryo1 MT1Cryo1 MT1Cryo1 LCACryo7* LCACryo9*

1 2 3 4 5 6 7

1.27 1.15 1.22 1.14 1.07 1.38 1.11

Å Å Å Å Å Å Å

“prior knowledge” strategy

composition 75% (30 μL) C4,a 25% w/v MPEG 2K, 100 mM KSCN,100 mM AAB 50/50 C3,a 18% w/v MPEG 2K, 100 mM AABb 50/50 C3,a 18% w/v MPEG 2K, 100 mM AABb 20/80

1

strategy b

C1, 10% PEG w/v 10K, 200 mM NaCl, 100 mM PCTP 80/20 crystallization

*database entries

high MW PEG instead of low MW PEG, different pH. crystal resolution

Same crystallization conditions MT1 (PDB entry 4DO8) 36% MPEG 2K, 450 mM NaCl, 90 mM KSCN, 100 mM imidazole HCl, pH 7.5 crystallization conditions use the same precipitant as cryo-formulation at the same pH (pH 7) crystallization conditions use the same precipitant as cryo-formulation with pH variation of the same buffer at a more basic pH

a

Cryomix C1 consists of 12.5% diethylene glycol + 12.5% MPD + 37.5% 1,2-propanediol + 12.5% DMSO; cryomix C3 consists of 12.5% diethylene glycol + 12.5% ethylene glycol + 12.5% MPD + 12.5% 1,2-propanediol + 12.5% DMSO + 12.5% glycerol + 12.5 mM 3-(-pyridinio)-1propanesulfonate (NDSB 201); cryomix C4 consists of 25% diethylene glycol + 12.5% ethylene glycol + 12.5% MPD + 12.5% 1,2-propanediol + 12.5% glycerol; cryomix C6 consists of 12.5% ethylene glycol + 25% MPD + 12.5% 1,2-propanediol + 12.5% DMSO + 12.5% glycerol. Percentages are v/v. It is mixed so that it represents 40% of the volume of the final cryoprotectant solution unless specified otherwise (Figure 1). bAAB is a linear buffer14 consisting of sodium acetate, ADA, bicine in the ratio 1:1:1 mixed as indicated by x/y from x% at pH 4 and y% at pH 9; similarly PCTP consists of sodium propionate, sodium cacodylate, and Bis-Tris-propane; mixed as indicated by x/y from x% at pH 4 and y% at pH 9.5. IM consists of mixed 1 M imidazole and 1 M D,L-malic acid. The buffer contribution represents 10% by volume of the final volume of the cryoprotectant solution. sessions, database cryo-formulations M9cryo5 and M9cryo7 were tested because successful in preserving a wide variety of crystals and similar in composition to M13cryo2. The success rate of new tests was better in the second synchrotron session and totally reliable on the third one with diffraction extending to between 2.7 and 2.4 Å resolution. The cryopreservation of crystals of a mutant of υ-Da2a, a kunitz domain protein from mamba venom can be used to illustrate a “prior knowledge” application where cryomixes C4 and C10 participate in the arena. A 2× precipitant is not possible, but the crystallization conditions do not require any cryoprotectant. This information was determined during a previous cryopreservation experiment with the three-finger fold muscarinic toxin MT1 from green mamba venom (PDB entry: 4DO8)17 that crystallized under identical conditions as the υ-Da2a mutant. The arena for the MT1 crystals yielded a “champion” consisting of the crystallization condition itself and a “challenger” MT1Cryo1 based on cryomix C4 (Table 1), which gave a cleaner background but slightly worse diffraction (1.9−2 Å compared

to 1.8 Å; not statistically significant). Exceptional results were obtained from all seven crystals tested (Table 1), but this time the challenger with three crystals, performed better that champion (1.07 Å compared to 1.15 Å; not a statistically significant battle; see Table 1). The prior knowledge tests also included a variation with less PEG taken from the database of premixed cryo-formulations giving the worse result with 1.38 Å resolution, while an increase in pH restores diffraction to 1.11 Å suggesting that a higher pH MT1Cryo1 might be an interesting variation that could help the challenger defend cryomix C4. The lower 1.38 Å resolution was mainly due to radiation damage, which allowed the structure to be solved automatically using radiation damage induced phasing (RIP).18 Long Soak Example. Formulations with the ability to preserve the crystals for extended periods of time are needed for soaking experiments to allow the introduction, removal or exchange of ligands in preformed crystals. The use described in Table 2 is for the removal of acetohydroxamic acid (AHA). As described in Devel et al.,19 AHA is a metalloprotease inhibitor that chelates the catalytic zinc. It is D



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Table 2. Long Duration Soaks crystallization

cryosolution

resolution (Å)

MMP12DL077

27% w/v PEG 10K, 100 mM glycine, pH 9.0

M12IM: 30% w/v MPEG2K, 10% v/v ethylene glycol, 200 mM imidazole malate pH 8.5

1.5

MMP12DL106

27% w/v PEG 10K, 100 mM Tris-HCl, pH 9.5

M12Cryo1: C2,a 10% w/v PEG 10K, 200 mM NaCl, 100 mM AAB 10/90.

1.8

MMP12LD837I

27% w/v PEG 10K, 200 mM imidazole malate, pH 8.5

M12Cryo7: C7,a 25% w/v MPEG 5K, 100 mM AAB 10/90.

1.63

MMP8DL110

25% w/v PEG 4K, 125 mM NaCl, 100 mM MES, pH 5.5

M8Cryo2: 5% v/v diethylene glycol, 10% v/v 1,2-propanediol, 10% v/v glycerol, 12% w/ v PEG10K, 13.7% w/v MPEG 2K, 25 mM MES, pH 5.5

1.6

MMP13DL110

17.5% w/v PEG 20K, 20 mM NaCl, 100 mM MES, pH 5.5

M13Cryo3: C3,a 10% w/v PEG 20K, 100 mM AABb 80/20.

2.5

example

soak length 5 min to remove AHA 24 h to remove AHA 5 min to remove AHA 24 h to remove AHA 30 s to remove AHA

a

Cryomix C2 consists of 25% diethylene glycol + 25% glycerol + 25% 1,2-propanediol. Cryomix C3 consists of 12.5% diethylene glycol + 12.5% ethylene glycol + 12.5% MPD + 12.5% 1,2-propanediol + 12.5% DMSO + 12.5% glycerol + 12.5 mM NDSB 201 Cryomix C7 consists of 12.5% diethylene glycol + 12.5% ethylene glycol + 25% 1,2-propanediol + 12.5% DMSO + 12.5% glycerol. % is v/v and the cryomix represents 40% of the volume of the final cryoprotectant solution (Figure 1). bAAB is a linear buffer14 consisting of sodium acetate, ADA, bicine in the ratio 1:1:1 mixed as indicated by x/y from x% at pH 4 and y% at pH 9. It represents 10% by volume of the final volume of the cryoprotectant solution.

of protein−protein complexes.23 The preponderance of PEGs ensures that the number of cases for which the standard cryoformulation (Figures 1 and 2) would require modification is relatively small. Selection Criteria. It can be argued that the only effective criterion for a good cryoprotectant solution is its ability to allow for the collection of complete high resolution data sets at a synchrotron radiation source. Unfortunately this criterion alone would be too slow to screen sufficient conditions to decide on a trend to follow and would be unsuitable to test most reasonable combinations, even for a single crystal form. An initial filter needs to be applied to overcome this problem. We have introduced the requirement that crystals should be stable for long periods of time in the solution. Since most crystals crack or dissolve in a few seconds, this first test is fast enough to be effective. This choice directs the evolution of the cryoprotectant mixtures toward balancing the solubilizing effect of the glycols with cryo-precipitants so combinations that neither crack crystals nor dissolve them are favored. Such balanced neutral solutions are likely to work broadly for a wide spectrum of proteins since the differences between crystals are taken care of by the modularity of the system. Thus the cryoprotectant combinations are effective on a wide range of protein crystals, without reformulation and irrespective of the crystallization conditions. Improving Resolution. Improved resolution is the result of better order in the crystal. This can be achieved by the wise use of a cryoprotectant solution, but we have had better success in introducing resolution enhancing additives in the crystallization step. The use of a dehydrating cryoprotectant solution can shock a crystal and force it into a better diffracting lattice.24 To avoid excessive mosaicity, the process must allow for reannealing. Without the supersaturated protein which maintains stability in the crystallization drop, crystals tend to melt in many cryoprotectant solutions that fail to compensate for the lack of mother-liquor. After rebalancing the cryoprotectant to prevent crystal melting by mixing cryoprotectant compounds with solubilizing and precipitating properties, the next step is has been to increase the concentrations of compounds, such as MPD, that have been used to enhance diffraction. The strategy of using MPD both as a cryoprotectant and to improve diffraction can be ascribed to the work on the

required at various concentrations to prevent protein degradation during crystallization. Since at high concentration it can be competitive with other inhibitors it needs to be removed. The soak time required for removal varies from one metalloprotease to another (up to 24 h for MMP-12). All soaks described in Table 2 successfully removed AHA as shown by X-ray diffraction studies.19 Data Collection. About 1000 samples were tested at synchrotron facilities, namely, at the ESRF (beamlines ID29, ID14-1, ID14-2, ID143, ID14-4, ID23-1, ID23-2, and BM30) in Grenoble and at the Soleil storage ring on beamline Proxima-1 in Saclay. More than ten proteins and more than 70 inhibitors and were analyzed over a period of three years, during which 2−6 different formulations of cryoprotectant solutions were tested for each protein or protein-complex during each session. Crystals were tested as soon as they were obtained as long as their size was believed suitable for data collection. When larger crystals became available, new tests were carried out on the bigger crystals using the cryoprotectant solution that had worked the best for the smaller crystals and other cryoprotectant solution optimized for other inhibitor complexes of the same or other proteins (prior knowledge path Figure 2). Data processing was carried out using the automated system available at the synchrotron facilities or on the laboratory computers.20,21

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RESULTS Modularity and Persistence. The result is an evolutive modular system that allows for mixing a large variety of different cryoprotectant combinations (Figure 1). Certain cryoprotectant combinations that gave good results for a particular protein, were found to be effective on various other proteins, often without any reformulation and irrespective of the crystallization conditions (Table 1). This may bare a relationship to the screening methodology, that starts from the use of the “Stura Screens”22 for the identification of the initial crystallization conditions, and introduces variations only later, and only when needed. Using this strategy, proteins that are easy to crystallize tend to do so under similar conditions. The Stura Screens subdivide the crystallization space into two classes of precipitants: PEGs and salts. Crystals obtained in one of the PEG conditions often give good results in a cryoprotectant solution with a high molecular weight PEG, while those grown in salt conditions, work well in cryosalts. In our screening, we have had greater success with PEGs, compared to high-salt crystallization conditions roughly in the same ratio (71% versus 27%) as reported for the crystallization E



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data collection trials is retained and used in subsequent data collection sessions and competed against new precipitantcryoprotectant-buffer recombinations. Quickly champion solutions emerge but since challengers are introduced at each data collection cycle new formulations will eventually give rise to new champions. Champions that were successful in cryoprotecting one or more crystal forms over several data collection sessions will face new challenges as the interest moves to new projects. Duplication is not needed in the champion competition method. Thus, solutions that do not work consistently are eliminated. Duplication could prevent unlucky and unfair elimination, but it is not useful as long as solutions containing the same core components persist in the competition arena. However, duplication is carried out to understand crystal-to-crystal variability, important in difficult cases (Table 1). Evolution of New Solutions. Evolution occurs often in response to new situations (Figure 2). If we consider the example of crystals that were found spontaneously in an eppendorf tube (h-MMP13) Table 1), we may realize that “champions” that were suitable for high PEG or salt conditions would be severely challenged. Indeed, on the first data collection session only 1 crystal out of 15 gave useful data (Table 1). The first successful “champion” (20% w/w MPEG2,000, 10% v/v MPEG-550, 10% v/v ethylene glycol, 90 mM imidazole malate, pH 6.0; PDB entry 4FU4 at 2.85 Å15) was eventually replaced by a database cryo-formulation evolved for a different project. We should note that in Figure 1, small molecular weight PEGs, like MPEG-550, are no longer represented. The database solution contains diethylene glycol instead of MPEG-500 with MPD, 1,2-propanediol and DMSO giving a resolution of 2.44 Å (PDB id 4FVL).15 This shows how components can disappear to be replaced by others. We may note that diethylene glycol (MW 106) instead of MPEG-550 is present in eight out of nine of the new champion solutions (Figure 1). The spreading of components over several solutions is due to the fact that new challengers are created by the recombination of successful characteristics found in other cryoprotecting solutions with those of the new champion. The spreading of diethylene glycol is also matched by a decrease in glycerol and ethylene glycol concentrations in the mixes. Glycerol remains present in eight out of nine of the current solutions and propanediol is found in all nine solutions. Evolutionary systems have a tendency to select neutral as well as the best variants. The failures of the evolutive system are countered by the prior knowledge optimization (Table 1; Figure 2) that allows challenges across projects for faster evolution of cryo-solutions and the optimization of cryosolutions specific to individual proteins.

guanine nucleotide-exchange factor complex, EF-Tu-Ts, where the resolution was extended from 5.0 to 2.5 Å.25 Our results also show that MPD works well in cryoprotectant solutions. When added in the cryo-soak step it is less likely to become effective in lattice stabilization than when used as an additive in the crystallization step. We have been able to achieve 1.2 Å with MPD as an additive, while without it the data were at only 1.8 Å. This may be explained by an MPD molecule at a crystal contact (PDB entry 3NDS27) something that may be harder to achieve systematically over the whole lattice in a regular manner during a short 5−30 s soak. In the present study, all cryoprotectant solutions with MPD were used in a single step soak, without transferring the crystals into successive cryosolutions, each with a greater precipitating power than the previous one, as practiced by the original investigators. Our system could also be used with multiple steps without modification, each step involving a solution with increasing MPD concentration. The selection procedure would require more crystals and would increase in complexity, but the final results could make it worth the effort. Several other post crystallization manipulations can increase the quality of the data that can be collected in house or at synchrotron sources such as dehydration and annealing.24 A procedure involving some form of dehydration, such as mixing a cryoprotectant solution with a higher concentration of precipitant or by introducing a cryo-precipitant component in the solution, is a good guideline. In this study, we have used unbuffered 80% saturated lithium sulfate at room temperature (2.5 M) with excellent results for most ammonium sulfate grown crystals. A single concentration of cryosalt, appears to be sufficient. The higher overall precipitating power of the 80% saturated lithium sulfate solution is effective in countering variations in protein solubility because of variations in buffer conditions, so that when crystals are transferred this solution they remain stable or show improvement. Similar results have been obtained with PEGs used at slightly higher concentrations than during crystallization. Cryosalts. Cryosalts have resisted very well in the arena. 80% saturated lithium sulfate (80SLS) has remained in the top position for many proteins crystallized in ammonium sulfate or other high ionic strength salts. In one case, crystals grown in 1 M NaCl were transferred into 80SLS in a single step without cracking. Lithium formate has done well and malonate has been also reported to be versatile.3 The possible dehydrating effect provided by cryosalts may contribute to improvements in resolution. In the case of 80% saturated lithium sulfate we have observed a substantial increase in the number of ordered sulfates in the electron density. The molecular weight of PEG component of the precipitant and the pH in the cryosolution mixes can be different from that used to grow the crystals (Table 1 and Figure 2). Also for cryosalts, the salt used in the cryosolution can be different to that used in the crystallization and the absence of buffer often gives excellent results although sometimes changing the pH can improve diffraction. Selection Strategy. Compared to the initial cryomixes in which some crystals cracked or dissolved or diffracted poorly, the final solutions behave well. Cryoprotectant solution evolution was carried out over many macromolecular crystals. The manner in which various cryo-solutions are used across projects is illustrated in Table 1. The strategy used to evolve the cryoprotectant mixes is a sort of champion competition method. The most successful cryoprotectant mix from previous

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DISCUSSION Optimization of a single cryoprotectant solution for a single protein can be a challenge if the whole range of possible single cryoprotectant compounds are tested at various precipitant concentrations. If we allow for some dehydration to optimize lattice contacts and a small variation in pH for the optimization of electrostatic interactions, the cryo-formulations that need to be mixed becomes to vast to screen. Yet, this is what our investigations suggest is the best approach (Figure 2). Faced with the inability to test everything, we must chose a method that can be optimized systematically in a limited number of steps and that can be useful from one protein to another. The champion-challenger approach is one such strategy. Only a F



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as an additive which in the cryoprotectant solution was increased to 15% MPD, providing extra precipitating power.28 Small MW PEG and MPD are not the only cryoprotectant molecules that can act as precipitants for the crystallization of proteins. Molecules, such as 1,2-propanediol, when used at high concentration can act as a stand-alone precipitants. Examples are E. coli L-rhanmulose-1-phosphate (mutant Q6Y-E192A, resolution 1.23 Å; see methods for PDB entry 2V9L)29 and a putative monooxygenase from Shewanella loihica (1.26 Å resolution; see methods for PDB entry 2RIL) where in both cases crystals were grown from 40% 1,2-propanediol. The current set of cryomixes could be tested as precipitants and using a similar selection strategy more effective cryoprecipitants mixes might evolve. Small molecule cryoprotectant compounds. The choice of small cryoprotectant compounds with the exclusion of slightly larger PEGs such as PEG 200 is based on the likelihood that the smaller compounds could diffuse in the crystal lattice faster than larger molecules. While the small MW PEGs are suitable for crystal soaks, the same is not true for the larger MW PEGs. In the study using crystals of house dust mite major allergen Der p 116,30 the crystals were stabilized in 45% w/v MPEG5000 or 2000 in 50 mM sodium acetate, pH 5.5 over several days. The high molecular weight PEG protects the crystals during flash-cooling but diffuse ice-rings are visible in the diffraction pattern (Figure 3a) with only a minor effect on data

limited numbers of tests are carried out on individual projects (Table 1), but the use of solutions across projects vastly expands the number of trials carried out with each cryomix (Figure 1). Over the vast number of crystals tested to date it has become clear glycerol nor any single compound cryoprotectant has performed better than any of the multicompound mixtures. The continued evolution of C4 (five mixed compounds) or any other cryomix is likely to be slower. The continued use of the recombination method used so far will lead to small compositional variations that are unlikely to have dramatic effects. The introduction of a new component could modify the system. When the additional component is introduced in the background of an established champion, it will carry a risk factor. If the new component is disliked by just one crystal form among all the ones tested, all cryomixes with that component will risk being eliminated in favor of those without that “risk factor”. The selection of unbuffered 80% saturated lithium sulfate (80SLS) as a single component cryosalt, rather than a mixture of salts shows that the “champion selection” strategy is not biased in favor of mixtures. Some cryosalts are poorly compatible with divalent cations present in the mother liquor. The presence of cations could result in salt crystals in the cryosolution. Lithium sulfate does not share this problem except when a high pH buffer is used. This may explain the preference for unbuffered 80SLS. Cryoprotectant Compounds in Structures. The ordered cryoprotectant molecules found in the refined structures can either participate in strengthening lattice contacts or become part of the solvent network. The reason for introducing cryoprotectant molecules is to destabilize the water network. The typical cryo-soak that last only a few seconds is ideal to achieve this. However, when cryomixes are used for long soak periods the possibility that they could act more like crystallization additives becomes real. The use of 80SLS leads to several sulfates becoming ordered in the electron density. These ions can participate in intermolecular salt-bridges that could increase the stability of the lattice. The strategy used to improve the diffraction of crystals the guanine nucleotideexchange factor complex, EF-Tu-Ts using successive long soaks in solvents containing higher MPD concentrations relied on crystal annealing. Annealing would allow crystals grown without structural cryoprotectant molecules to incorporate them in the lattice. The effect on crystal quality would in some cases result in improved resolution. Heavy atom soaking often leads to loss of order, but not always. The uranyl molecule in PDB entry 3MKO was introduced by soaking, leading to an improvement in resolution over the native crystals.26 An increase in resolution because of the incorporation of a structural cryoprotectant molecule during a cryo-soak of any length is an unlikely event, but the use of cryomixes instead of individual components as a crystallization additive could have certain benefits. In our structure at 1.2 Å, MPD was used as an additive (5% v/v) in the crystallization.27 Of the 30 entries in the PDB database at 1.2 Å or better, with ordered MPD molecules, in almost all cases MPD was used as a precipitant or an additive (Search statistics 1159 entries resolution 1.2 Å or better, 651 entries with ordered MPD). In only 2 cases, it was introduced during the cryo-soak. The concentrations required are compatible with what the mixes can provide. The crystallization conditions for atomic resolution structure of prostaglandin D2 11-ketoreductase consisted of 25% PEG 4000 with 2.5% MPD

Figure 3. High molecular weight polyethylene glycol as a cryoprotectant. (a) Diffraction pattern for crystals used to solve the structure of the C2 crystal form of mite allergen Der P1 (PDB entry 3F5V). The soaking allows protection but diffuse ice rings are still evident. (b) Crystals of the muscarinic toxin MT1 grown in high molecular weight polyethylene glycol give a diffraction pattern without diffuse ice rings.

quality (see P21212 (PDB code 2AS8)16 and C2 (PDB code 3F5V).30 However, if instead of long-term soaking the crystals are grown directly in MPEG-2000 (MT1, PDB code 3FEV and υ-Da2a; Table 1) the resultant diffraction pattern shows no diffuse ice-rings (Figure 3b). This suggests that during the soak the high MW PEG cannot penetrate the crystal lattice. However, during crystal growth PEG molecules are trapped within the lattice and provide protection against the formation of ice nanocrystals when flash-cooled. This observation is important as it guides toward the use of small cryoprotectants compounds for cryo-soaks. Ligand Solubilization. The presence of dimethyl sulfoxide (DMSO) in six out of nine solutions and the fact that crystals last longer in these mixed cryoprotectant solutions compared to solutions prepared from single compound with added DMSO, allows for the soaking in or out of poorly water-soluble G



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provided for use in our laboratory in the interest of quality control to ensure that it corresponds well to the kit that was developed. This transfer of technology conforms with the mission of the CEA: “from research to industry”. The authors declare no competing financial interest.

inhibitors for over an extended time period (Table 2). With or without DMSO, the longevity of crystals in the cryoprotectant solution is an auburn for all postcrystallization interventions such as heavy atom or ligand soaks or back-soaks. Other methods to be considered to increase longevity is the use of gelled media that has been used as a means to strengthen crystals and facilitate cryocooling.31,32 Future Developments. The loss of diffraction due to radiation-damage obtained with υ-Da2a crystals and LCAcryo7 suggests that variations in cryo-formulation can influence the resilience of crystals in the X-ray beam. Better understanding this relationship could lead to developments that would identify compounds that may improve or decrease crystal longevity at synchrotron radiation sources. Those with shortened lifespans could be useful for RIP phasing,18 while those that survive longer could provide the best data. It is important to note that the current mixtures to not comprehensively include all valuable cryoprotecting compounds. Dioxane and dimethylene glycol (2,3-butanediol) are two notable omissions. Dioxane is also common additive in crystallization33 and dimethylene glycol has already shown its value as a glycerol substitute.34

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ACKNOWLEDGMENTS We are grateful to the ESRF and SOLEIL for beam time and to their staff for assistance and to various colleagues that have provided proteins for the crystallization and cryopreservation studies.

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(1) Kwong, P. D.; Liu, Y. Use of cryoprotectants in combination with immiscible oils for flash cooling macromolecular crystals. J. Appl. Crystallogr. 1999, 32, 102−105. (2) Rubinson, K. A.; Ladner, J. E.; Tordova, M.; Gilliland, G. L. Cryosalts: Suppression of ice formation in macromolecular crystallography. Acta Crystallogr., Sect. D 2000, 56, 996−1001. (3) Holyoak, T.; Fenn, T. D.; Wilson, M. A.; Moulin, A. G.; Ringe, D.; Petsko, G. A. Malonate: A versatile cryoprotectant and stabilizing solution for salt-grown macromolecular crystals. Acta Crystallogr., Sect. D 2003, 59, 2356−2358. (4) Teng, T.-Y.; Moffat, K. Cooling rates during flash cooling. J. Appl. Crystallogr. 1998, 31, 252−257. (5) Kriminski, S.; Kazmierczak, M.; Thorne, R. E. Heat transfer from protein crystals: implications for flash-cooling and X-ray beam heating. Acta Crystallogr,. Sect. D 2003, 59, 697−708. (6) Garman, E. F.; Mitchell, E. P. Glycerol concentrations required for cryoprotection of 50 typical protein crystallization solutions. J. Appl. Crystallogr. 1996, 29, 584−587. (7) Shah, B. N.; Chinte, U.; Tomanicek, J.; Hanson, B. L.; Schall, C. A. Flash cooling protein crystals: Estimate of cryoprotectant concentration using thermal properties. Cryst. Growth Des. 2011, 11, 1493−1501. (8) Vera, L.; Czarny, B.; Georgiadis, D.; Dive, V.; Stura, E. A. Practical use of glycerol in protein crystallization. Cryst. Growth Des. 2011, 11, 2755−2762. (9) Anand, K.; Pal, D.; Hilgenfeld, R. An overview on 2-methyl-2,4pentanediol in crystallization and in crystals of biological macromolecules. Acta Crystallogr., Sect. D 2002, 58, 1722−1728. (10) Tran, T. T.; Sorel, I.; Lewit-Bentley, A. Statistical experimental design of protein crystallization screening revisited. Acta Crystallogr., Sect. D 2004, 60, 1562−1568. (11) Stura, E. A. Strategy 3: Reverse screening. In Crystallization of Proteins: Techniques, Strategies and Tips. A Laboratory Manual; Bergfors, T.; Ed.; International University Line: La Jolla, CA, 1998; pp 113−124. (12) Stura, E. A. Seeding Techniques. In Crystallization of Nucleic Acids and Proteins: a practical approach Second ed., Ducruix, A., Giegé, G., Eds. Oxford University Press: Oxford, U.K., 1999; pp 177−208. (13) Stura, E. A.; Gleichman, T. Soaking of Crystals. In Crystallization of Nucleic Acids and Proteins: A Practical Approach, 2nd ed.; Ducruix, A., Giegé, G., Eds.; Oxford University Press, 1999; pp 365−390. (14) Newman, J. Novel buffer systems for macromolecular crystallization. Acta Crystallogr., Sect. D 2004, 60, 610−612. (15) Stura, E. A.; Visse, R.; Yiotakis, A.; Acharya, R.; Murphy, G.; Dive, V.; Nagase, H. Crystal structures of full-length human collagenase 3 (MMP-13) with peptides in the active site defines an exosite in the catalytic domain. Manuscript in preparation (PDB codes 4FU4, 4FVL, 4G0D). (16) De Halleux, S.; Stura, E. A.; VanderElst, L.; Carlier, V.; Jacquemin, M.; Saint-Remy, J.-M. Three-dimensional structure and IgE-binding properties of mature fully active Der P 1, a clinically relevant major allergen. J. Allergy Clin. Immunol. 2006, 11, 571−576. (17) Fruchart-Gaillard, C.; Mourier, G.; Blanchet, G.; Vera, L.; Ménez, R.; Marcon, E.; Stura, E. A.; Servent, D. Engineering of three-

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CONCLUSION Our approach is relatively simple since we replace single component cryoprotectant compounds with a mixture that provides the same protection, but with better results since the formulations do not require an increase in precipitant concentration to stabilize crystals. Until recently glycerol could have been considered the universal winning cryosolution if tolerated by the crystals, since at 32% w/v it almost always inhibits ice formation. Each cryomix at 2.5×, can be considered the replacement of 80% w/v glycerol (see Figure 2 prior knowledge). Since the solubilization effect of the glycols is dose-dependent, it is beneficial to use them sparingly in cryoformulations.7 The mixed solutions contain smaller amounts of these potentially damaging additives so that it is less likely to see crystals melt or lose their crystalline order. In addition, because the mixed compositions contain a balanced level of precipitating compounds they are also less likely to lead to the cracking of crystals. The strategy for protein crystal cryoprotection presented in the paper is the use of the “champion-challenge” approach to arrive at a set of mixtures that have a higher success rate than just single components. The methodology used to evolve the mixed component cryoprotectants is more important than the composition of the current solutions. Adoption of the methodology would allow for new and interesting molecules to become part of better performing mixed formulations.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors have no personal financial interest in the agreement stipulated between CEA Life Sciences Division, Saclay, (France) and Molecular Dimensions Ltd. (U.K.) for the marketing and sales of the kit formulated in our laboratory under the name CryoProtX, for which Molecular Dimensions has negotiated an exclusive license to manufacture and distribute the developed formulation of cryoprotectant solutions. The authors acknowledge the continued relationship with Molecular Dimensions where free samples are to be H



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finger fold toxins creates ligands with original pharmacological profiles for muscarinic and adrenergic receptors. Plos One 2012, 7, e39166. (18) Zwart, P. H.; Banumathi, S.; Dauter, M.; Dauter, Z. Radiationdamage-induced phasing with anomalous scattering: substructure solution and phasing. Acta Crystallogr., Sect. D 2004, 60, 1958−1963. (19) Devel, L.; Beau, F.; Amoura, M.; Vera, L.; Cassar-Lajeunesse, E.; Garcia, S.; Czarny, B.; Stura, E. A.; Dive, V. Simple pseudo dipeptides with a P2′ glutamate: A novel inhibitor family of MMPs and other metzincins. J. Biol. Chem. 2012, 287, 26647−26656. (20) Leslie, A. G. W.; Powell, H. R.; Winter, G.; Svensson, O.; Spruce, D.; McSweeney, S.; Love, D.; Kinder, S.; Duke, E.; Nave, C. Automation of the collection and processing of X-ray diffraction dataA generic approach. Acta Crystallogr., Sect. D 2002, 58, 1924− 1928. (21) Kabsch, W. XDS. Acta Crystallogr., Sect. D 2010, 66, 125−132. (22) Stura, E. A.; Nemerow, G. R.; Wilson, I. A. Strategies in the crystallization of glycoproteins and protein Complexes. J. Cryst. Growth 1992, 122, 273−285. (23) Radaev, S.; Sun, P. D. Crystallization of protein−protein complexes. J. Appl. Crystallogr. 2002, 35, 674−676. (24) Heras, B.; Martin, J. L. Post-crystallization treatments for improving diffraction quality of protein crystals. Acta Crystallogr., Sect. D 2005, 61, 1173−1180. (25) Schick, B.; Jurnak, F. Extension of the diffraction resolution of crystals. Acta Crystallogr., Sect. D 1994, 50, 563−568. (26) Igonet, S.; Vaney, M. C.; Vonhrein, C.; Bricogne, G.; Stura, E. A.; Hengartner, H.; Eschli, B.; Rey, F. A. X-ray structure of the arenavirus glycoprotein GP2 in its postfusion hairpin conformation. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 19967−19972. (27) Gross, G.; Gallopin, M.; Vandame, M.; Couprie, J.; Stura, E.; Zinn-Justin, S.; Drevet, P. Conformational exchange is critical for the productivity of an oxidative folding intermediate with buried free cysteines. J. Mol. Biol. 2010, 403, 299−312. (28) Lovering, A. L.; Ride, J. P.; Bunce, C. M.; Desmond, J. C.; Cummings, S. M.; White, S. A. Crystal structures of prostaglandin D(2) 11-ketoreductase (AKR1C3) in complex with the nonsteroidal anti-inflammatory drugs flufenamic acid and indomethacin. Cancer Res. 2004, 64, 1802−1810. (29) Grueninger, D.; Treiber, N.; Ziegler, M. O.; Koetter, J. W.; Schulze, M. S.; Schulz, G. E. Designed protein−protein association. Science 2008, 319, 206−209. (30) Chruszcz, M.; Chapman, M. D.; Vailes, L. D.; Stura, E. A.; SaintRemy, J. M.; Minor, W.; Pomés, A. Crystal structures of mite allergens Der f 1 and Der p 1 reveal differences in surface-exposed residues that may influence antibody binding. J. Mol. Biol. 2009, 386, 520−530. (31) Gavira, J. A.; Garcia-Ruiz, J. M. Agarose as crystallisation media for proteins II: Trapping of gel fibres into the crystals. Acta Crystallogr., Sect. D 2002, 58, 1653−1656. (32) Bonnefond, L.; Schellenberger, P.; Basquin, J.; Demangeat, G.; Ritzenthaler, C.; Chênevert, R.; Balg, C.; Frugier, M.; RudingerThirion, J.; Giegé, R.; Lorber, B.; Sauter, C. Exploiting protein engineering and crystal polymorphism for successful X-ray structure determination. Cryst. Growth Des. 2011, 11, 4334−4343. (33) Ménétrey, J.; Perderiset, M.; Cicolari, J.; Houdusse, A.; Stura, E. A. Improving diffraction from 3 to 2 Å for a complex between a small GTPase and its effector by analysis of crystal contacts and use of reverse screening. Cryst. Growth Des. 2007, 7, 2140−2146. (34) McFerrin, M. B.; Snell, E. H. The development and application of a method to quantify the quality of cryoprotectant solutions using standard area-detector X-ray images. J. Appl. Crystallogr. 2002, 35, 538−545.

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Strategies for Protein Cryocrystallography - Crystal Growth & Design

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