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A new design of protein crystallization plates to expand concentration screening space in cross-diffusion microbatch and microbatch methods Chen Dong, Chen-Yan Zhang, Yang-Yang Liu, Ren-Bin Zhou, Qing-Di Cheng, and Da-Chuan Yin Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.5b00702 • Publication Date (Web): 06 Jan 2016 Downloaded from http://pubs.acs.org on January 7, 2016

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Crystal Growth & Design

A new design of protein crystallization plates to expand concentration screening space in crossdiffusion microbatch and microbatch methods Chen Dong†, Chen-Yan Zhang†, Yang-Yang Liu, Ren-Bin Zhou, Qing-Di Cheng, Da-Chuan Yin* †

C.D. and C.Y.Z. contributed equally to this work.

Institute for Special Environmental Biophysics, Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi’an 710072, Shaanxi, PR China

Email of the corresponding author: Da-Chuan Yin: [email protected]

KEYWORDS：：protein crystallization, cross-diffusion microbatch, microbatch, crystallization plate, sitting-drop vapor diffusion method, protein concentration, crystallization screening

ACS Paragon Plus Environment

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Abstract: Protein crystallizes at specific conditions (correct precipitants, appropriate concentrations of protein and precipitants, suitable pH and correct temperature, etc.). If the conditions are not appropriate, crystallization will not occur. In protein crystallization screening, the target protein is mixed one by one with many chemical agents and then incubated at a set temperature. If the concentrations of the chemical agents and the target proteins are not in a range suitable for crystallization, the crystallization will not occur. To expand the concentration screening space, we propose in this paper a new design of protein crystallization plates for crossdiffusion microbatch and microbatch methods. The new plates have 96 units corresponding to the conditions of the commercial screening kits, and each unit contains four wells for holding the crystallization droplets. By dispensing crystallization droplets to the four wells at different volume ratios of protein to precipitant solutions, we can obtain four different initial concentrations for each unit, thus a wider concentration range can be screened. The comparison between the screening performance of the new plates and the traditional sitting-drop vapor diffusion plate showed that the new design of the plates exhibited significantly improved results in obtaining more crystallization conditions.


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Introduction Finding crystallization agents (or precipitants) for proteins is crucial when X-ray protein crystallography 1, 2, which is the most widely applied method for obtaining structural information in proteins, is used

3-5

. Typically, protein in an aqueous solution needs some chemical agents to

help to adjust the interactions between the protein molecules so that the crystallization can happen 6, 7. Therefore, these chemical agents are crucial to the process. However, currently there is no theoretical method to identify the correct chemical agents to help crystallize the target protein. To determine what chemical agents are suitable precipitants, a trial and error test, in which different chemical agents are mixed one by one with the target protein to see which one can aid in crystallization, is needed

8-10

. This process is often referred to as the crystallization

screening process 11. In a crystallization screening experiment, the screening space of crystallization parameters should be in appropriate ranges (preferably wider ranges). The parameters (or variables) needed to be considered are usually the precipitant type 12, the concentrations of protein and precipitants 13-15

, the pH value

16

, and the temperature

9, 17-20

. When a crystallization screening kit, which

comprises different recipes of chemical agents, is chosen for the screening, the important parameters that need to be well controlled are: the concentrations of the protein and the precipitants (from the screening kit), pH and the temperature 21, 22. In the case of selecting concentrations for precipitants and protein, the methods that can vary the concentrations of the precipitants and the protein already exist. For example, the most widely used method, vapor diffusion, can vary from an initial concentration to a maximum concentration which is largely determined by the reservoir concentration


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. A number of other

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methods, such as free-interface diffusion (FD) microbatch methods

31

27-29

, desiccation

30

24

, counter-diffusion (CD)

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24

, dialysis

25, 26

,

and the recently reported cross-diffusion microbatch (CDM)

can also vary the concentration in a wide range. However, the screening space of all

of these methods is still limited to some extent. For example, the vapor diffusion method, the most widely used method, has a limited concentration range. Though it is possible to extend the concentration range from a lower initial concentration to a higher end concentration by manipulating the initial concentration of the droplets automatically or manually, the concentration space screened is still limited. When the first screening is not satisfactory, it is a common practice in many laboratories to vary the volume ratio of protein and precipitants solutions

32-34

to expand the screening space. However, it is not always convenient to do so. To

make it easier and more practically applicable, we here propose a new design for crystallization plates that can enable multiple initial concentrations in one crystallization plate. The principal idea of this design is to simultaneously realize multiple initial concentrations for each of the 96 screening conditions so that no further volume ratio adjustment is required. Two types of crystallization plates can be prepared according to this idea: 1) Type 1 plate comprises 96 units corresponding to the 96 screening conditions. Each unit comprises four wells. All 96×4 wells share one common space so that vapor diffusion can happen among all of the wells. Since its crystallization method follows CDM method, this plate is named as CDM plate in this paper. 2) Type 2 plate is similar like Type 1 (CDM) plate, the only difference is that, each of the 96 units is sealed separately and the four wells in each unit share a common space. This type of plate follows microbatch crystallization method, so it is named as M plate in this paper. Compared with the commercial plates, there is no reservoir well for the two types of newly-designed plates.


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We performed crystallization screening experiments using the two types of new plates, and compared them with the conventional sitting-drop vapor diffusion method using an Intelli-Plate 96-2. The results showed that the combination of the new designed plates with commercial crystallization cocktails can help to identify more crystallization conditions.


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Materials and methods Materials Chemicals and consumables. Twelve commercial proteins were utilized. Detailed information on the proteins is listed in Table 1. All of the proteins were used directly without further purification. Sodium chloride was obtained from MP Biomedicals Company (Santa Ana, USA), sodium acetate and HEPES sodium were purchased from Beijing Chemical Factory (Beijing, People’s Republic of China). The crystallization screening kit IndexTM (catalogue No. HR2-144) and the Crystal Clear Tape (catalogue No. HR4-506) were from Hampton Research Corporation (Aliso Viejo, USA).


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Table 1. The buffers and initial concentrations for different proteins

Proteins

Abbr.

Catalogue

Buffer

Initial concentration (before mixing) (mg/ml)

lysozyme

lys.

100940

0.1 M NaAc, pH 4.6

20.0

proteinase K

prk.

P6556

25 mM HEPES, pH 7.0

20.0

catalase

cat.

C40

25 mM HEPES, pH 7.0

20.0

α-chymotrypsinogen A chy. type II

C4879

25 mM HEPES, pH 7.0

20.0

glucose isomerase

HR7

25 mM HEPES, pH 7.0

10.0

concanavalin A type VI con.

L7647

25 mM HEPES, pH 7.0

20.0

myoglobin

myo.

M1882

25 mM HEPES, pH 7.0

20.0

cellulase

cel.

C0615

25 mM HEPES, pH 7.0

20.0

ribonuclease A type I

rib.

R4875

25 mM HEPES, pH 7.0

20.0

papain

pap.

P3125

25 mM HEPES, pH 7.0

20.0

hemoglobin

hem.

H2625

25 mM HEPES, pH 7.0

20.0

subtilisin A type VIII

sub.

P5380

25 mM HEPES, pH 7.0

20.0

glu.


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Crystallization plates. Three types of crystallization plates were used in this study. The first type was designed for the CDM method, and it is referred to as CDM plate 96-4 (abbreviated as CDM plate) in this paper. The second type was designed for microbatch method, and it is referred to as microbatch plate 96-4 (abbreviated as M plate). The third type was commercial Intelli-plate 96-2 (Art Robbins Instruments, Sunnyvale, CA) for the sitting drop vapor diffusion (SDVD) plate. Fig. 1 shows the schematics of the three types of plates. The newly designed crystallization plates consist of 96 units, which are arranged in a manner compatible with the Society for Biomolecular Sciences (SBS) Standard suitable for the automated systems used for crystallization trials. The 96 units correspond to the 96 chemical agents from the crystallization screening kits. Each unit contains four wells for holding mixtures of the same chemical agents and protein, but with different volume ratios to create four different initial concentrations. The crystallization plates are made in two types. Type 1 (CDM plate) is designed for the CDM method, in which all of the wells are sealed in one common space so that any volatile matter can diffuse from one droplet to any other droplets (Fig. 1 a). Type 2 (M plate) is designed for the microbatch method, in which all 96 units are sealed separately, but the four wells in one unit are in one common space (Fig. 1b). Both CDM and M plates are water permeable so that the water vapor can slowly diffuse out from the plate, and thus the aqueous droplets of crystallization solution can be concentrated against crystallization time to create supersaturation and consequently induce crystallization. The SDVD plate, which is widely utilized in most laboratories, was used as control. Each plate contains 96 units, and each unit contains two wells for sitting-drops and one well for reservoir


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solution (Fig. 1c). Due to the coexistence of the reservoir and the crystallization droplets in one sealed space, vapor diffusion from the crystallization droplet (the initial precipitant concentration is usually one second of that in the reservoir) to the reservoir will happen so that the concentration of the crystallization droplets will increase to a level when the whole space reached an equilibrium.


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Figure 1. Crystallization plates used in this study. (a) CDM plate. It contains 96 units, and each unit consists of four wells. All 96×4 wells are in one common space so that any evaporated species can diffuse to any other droplets. (b) M plate. It contains 96 units, and each unit contains four wells. The units are separated but the four wells in one unit share the same common space. (c) SDVD plate. It contains 96 units. Each unit consists of two sitting-drop wells and one reservoir well.


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Methods. To compare the crystallization results using different crystallization plates, we conducted a screening experiment using the three plates for all 12 proteins. All of the experimental results were analyzed statistically to obtain a reliable comparison result. The proteins were dissolved in their corresponding buffers at the initial concentrations to obtain the protein solutions (the buffers and the initial concentrations for the proteins are listed in Table 1). These protein solutions were ready to use after centrifuging at 12,000 rpm for 15 min. For all three types of crystallization plates, the 96 units correspond to the 96 precipitant conditions of the IndexTM crystallization kits. In the cases of CDM and M plates, the four wells in each unit will be dispensed with the same volume of precipitant solution, but with different volumes against the protein solution. The volumes of protein and precipitant solutions (volume of protein solution + volume of precipitant solution) used for the four wells were: 0.5 µl + 1.0 µl, 1.0 µl + 1.0 µl, 1.5 µl + 1.0 µl, and 2.0 µl + 1.0 µl. In the case of the SDVD Plate, the crystallization procedure followed the standard SDVD method, and the volumes of the protein and the precipitant solutions for preparing the crystallization droplet were 1.0 µl + 1.0 µl. The volume of the reservoir was 80 µl. Crystal Gryphon (Art Robbins Instruments, Sunnyvale, CA) was used to set up crystallization trials. After setting up the crystallization trials using the crystallization robot, the crystallization plates were sealed using the Crystal Clear Tape and immediately placed into a temperature controlled chamber for incubation, which was controlled to a stable temperature within 0.1 K by circulating temperature controlled water from a water bath (Polyscience, Niles, USA). The temperature was 293 K, and the incubation time was 48 hours. The images of the droplets in each crystallization


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plate were captured using the automated crystal image reader (Xtal-Quest Inc., Beijing, People’s Republic of China) after incubation. All data and figures were analyzed using GraphPad Prism software (GraphPad Software Company, La Jolla, CA).


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Results CDM and M plates showed similar tendency in crystallization screening performance against the volume ratios of protein solution to precipitant solution. We examined the crystallization screening performance of the two new plates (CDM and M plates) at four different volume ratios (0.5 µl : 1.0 µl, 1.0 µl : 1.0 µl, 1.5 µl : 1.0 µl, and 2.0 µl : 1.0 µl) of protein solution to precipitant solution. Fig. 2 shows the number of crystallization screening hits (hits were defined as crystallization conditions that yielded crystals visible under the image reader) at the four volume ratios for the 12 tested proteins. It can be perceived that the number of crystallization screening hits showed similar developments (Fig. 2 a1 and b1) against the volume ratio for the two new plates, and the total number of screening hits for the two plates is comparable to each other. By normalizing and averaging the numbers of screening hits of all tested proteins for the two plates at different volume ratios, we can obtain the trend of average normalized screening hits against the volume ratio. Because the volume ratio represents the concentration level, the trend of number of screening hits against concentration can be obtained (Figs. 2 a2 and b2). From the figures (Figs. 2 a2 and b2), we can observe a clear trend in the increase in the number of screening hits against the volume ratio (i.e., against the protein concentration). This result indicates that, in the tested range of concentration, higher protein concentration is favorable for obtaining crystals for most of the tested proteins. To statistically verify the trend, we applied a one-way ANOVA test on the number of crystallization screening hits at different volume ratios. The results confirmed that the increasing


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trend of number of hits against protein concentration does exist in the tested range of concentration (Figures 2 a2 and b2).


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Figure 2. Number of crystallization hits at different volume ratios of protein and precipitant solutions. (a1) The number of crystallization screening hits using the CDM plate. (a2) The averaged normalized screening hits at different volume ratios. **P＜0.01, *P＜0.5. error bar: mean ± SEM, n =12; (b1) The number of crystallization screening hits using the M plate; (b2) The averaged normalized screening hits at different volume ratios. *P＜0.5. error bar: mean ± SEM, n =12. SEM: standard error of the mean. n: number of sample. Value of P represent reliability of result, *P＜0.5 indicates the difference are significant, **P＜0.01 indicates the difference are highly significant.


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CDM and M vs. SDVD plates. To determine if the crystallization screening performance of the two new plates improved when compared with the traditional SDVD plates, we compared the screening results using the three different plates. Prior to comparison, we first removed the redundancy that occurs in both the CDM and M plates, i.e., that units exist in which two or more wells yielded crystals. Fig. 3 shows several examples of such redundancy. The redundancy has been removed, i.e., any hits in each of the four wells in one unit count as one hit. Note that the size of the droplets seems to be different even though the initial volume ratio was the same (like in the row 0.5 : 1.0 in Fig. 3). There are two possible reasons for this observation. 1) the depth of the wells are different. The well depth of M plate is deeper than that of CDM plate. When the droplets were dispensed (and attached) to the side of the wells, the droplets will look smaller even if the actual volume is the same; 2) Since the chemical compositions for the droplets are different, the vapor pressure for each droplet will be different, resulting in different evaporation rate for different droplets. Hence the actual volume would be different after 2-days’ vapor diffusion (evaporation or condensation).


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Figure 3. Examples of redundancy in crystallization screening using CDM and M plates. The droplet images in this figure show the crystallization results of lysozyme in unit A8 using a CDM plate, lysozyme in unit G11 using an M plate, proteinase K in unit A12 using a CDM plate, and proteinase K in unit B3 using an M plate at different volume ratios of protein solution to precipitant solution. It can be seen that, in the same unit, there might be more than one droplet that yields crystals. The redundancy was removed by counting the number of units that yielded crystals, i.e., any wells that yielded crystals in the same one unit count as one hit.


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After removing the redundancy, the comparisons between different crystallization plates are summarized in Fig. 4. Figs. 4 a1-a2, b1-b2, and c1-c2 are comparisons between CDM and SDVD plates, M and SDVD plates, and CDM and M plates, respectively. It can be seen from the comparisons shown in Figs. 4 a2 and b2 that both CDM and M plates are helpful in increasing crystallization chances when compared to the traditional SDVD plate. The improvement in screening performance was significant for both CDM and M plates, as verified by the t-test method. Compared with the SDVD plate, the average normalized screening hits were increased by 249% and 212% by using CDM plate and M plate, respectively. We also compared the screening performance between the CDM and the M plates (Figs. 4 c1 and c2). It can be seen that the screening performance of these two plates is similar, and any difference is not significant (Fig. 4 c2).


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Figure 4. Comparisons of the number of crystallization screening hits using different crystallization plates (SDVD Plate, M Plate, and CDM Plate). The number of crystallization screening hits for the M and CDM plates is without redundancy, i.e., hits in one unit count as one hit. (a1) Comparisons of the number of screening hits between CDM and SDVD Plates. (a2) The averaged normalized screening hits on the CDM plate compared to the SDVD Plate, **P＜0.01, error bar: mean ± SEM, n =12. (b1) Comparison of the number of screening hits between M and SDVD Plates. (b2) The averaged normalized screening hits on the M plate compared to the SDVD Plate, **P＜0.01, error bar: mean ± SEM, n =12. (c1) Comparison of the number of screening hits between CDM and M plates. (c2) The averaged normalized screening hits on the CDM plate compared to the M Plate. error bar: mean ± SEM, n =12.


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Combining CDM and M plates to increase the screening performance. As shown in Fig. 3, except for the observed redundancy in CDM plates and M plates, we can also note that different initial volume ratios may yield completely different crystallization results, even though the same chemical agents are used. For example, lysozyme did not crystallize when the ratio of protein solution to precipitant solution was 0.5 : 1.0, but it crystalized when the ratio increased above 1.0 : 1.0, in unit A8 (0.1 M Sodium acetate trihydrate, pH 4.5, 3.0 M Sodium chloride) using the CDM plate (Fig. 3). This phenomenon indicates that different crystallization plates may find different crystallization conditions. Tables 2 and 3 summarize the number of crystallization conditions found only by either the CDM or the SDVD plates and only by either the M or the SDVD plates. It can be seen that by using different plates, we always have the opportunity to find new crystallization conditions. From Tables 2 and 3, it can also be observed that both CDM and M plates can find more new conditions than those found only by the SDVD plate. Fig. 5 shows the comparison results of the crystallization conditions found only by the CDM and M plates compared to those found only by the SDVD plate. The statistical analysis (t-test) showed that the number of crystallization conditions found only by either the CDM or the M plate was dramatically higher than that found only by the SDVD plate.


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Table 2. Crystallization conditions found only by either the CDM plate or the SDVD plate.

Proteins

Number of crystallization conditions Number of crystallization conditions found only by the CDM plate found only by the SDVD plate

lys.

31

1

prk.

13

1

cat.

29

1

chy.

8

12

glu.

29

2

con.

9

7

myo.

8

1

cel.

2

3

rib.

3

1

pap.

6

1

hem.

6

1

sub.

5

0


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Table 3. Crystallization conditions found only by either the M plate or the SDVD plate.

Proteins

Number of crystallization conditions Number of crystallization conditions found only by the M plate found only by the SDVD plate

lys.

19

2

prk.

9

9

cat.

32

1

chy.

39

6

glu.

25

5

con.

8

3

myo.

12

1

cel.

2

1

rib.

6

0

pap.

4

1

hem.

5

1

sub.

4

0


22

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Figure 5. Comparison of the number of crystallization conditions found by only one plate. (a) Comparison of the number of crystallization conditions found only by CDM plate and SDVD plate. (b) Comparison of the number of crystallization conditions found only by M plate and SDVD plate. *P＜0.05, **P＜0.01. Error bar: mean ± SEM. n=12.


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From the above results, we anticipate that combining the crystallization plates may be helpful in increasing the probability of obtaining crystals. To confirm this theory, we combined the two new crystallization plates (CDM and M plates) and compared the combined crystallization screening results (without redundancy) with those of the SDVD plate. The method of combining the crystallization screening results is to sum up all of the crystallization conditions, but remove the redundancy, i.e., any of the CDM and M plates succeeds to yield crystals at a precipitant condition will be counted as 1 hit and summed up to the total number of hits, but when both CDM and M plates yield protein crystals using the same serial number of precipitants from the screening kit, the crystallization result will be counted as 1 hit. Fig. 6 shows the comparison results. It can be seen that all 12 tested proteins showed the same tendency with the combination of CDM and M plates to find more crystallization conditions (Fig. 6a). The statistical analysis of the average normalized number of screening hits showed a dramatic increase (by 521%) by using the combination when compared to using a single SDVD plate.


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Figure 6. Comparison of the number of crystallization screening hits using the combination of the two new plates versus the SDVD plate. (a) Number of screening hits using the combination of the two new plates (CDM and M plates), and using the SDVD plate. (b) Comparison of the averaged normalized crystallization screening hits using the combination and the SDVD plate. **P＜0.01. Error bar: mean ± SEM. n=12.


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Discussions The above comparisons showed clearly that the two new crystallization plates can significantly increase the probability of obtaining crystals. To gain a better understanding of the phenomenon, we roughly predicted the possible paths of the concentration evolution in the phase diagrams for different cases. The actual vapor diffusion process among the droplets (either in M or in CDM plate) can be very complicated, and may be practically not possible to draw a strictly correct path in the phase diagram for such process. To roughly estimate the vapor diffusion process, it is necessary to give some assumptions for the conditions of the solution to simplify the situation so that the vapor diffusion can be predictable in an ideal condition. The assumptions for the initial conditions are as follows: 1) No crystallization, precipitation, or chemical reaction occurs in the droplets. 2) Water is the only volatile component for all droplets. 3) There is no solute left on the well bottom when the droplet is shrinking during concentration. 4) The solute can distribute in the droplets homogeneously at all time so that the concentration in the solution is always homogeneous. Under the conditions with the above assumptions, the solvent (i.e., water) will evaporate and diffuse among the droplets and finally reach an equilibrium following the Raoult’s law. The evaporation of water from a droplet (or condensation of water into a droplet) will lead to the change in the concentration. The concentration variation path of the solution in the phase diagram will follow a linear line, because as mentioned in the assumption, only water is volatile in the droplets, and thus the mass ratio of protein to precipitant should be constant.


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According to the above theoretical considerations, we drew the phase diagrams for different cases when the crystallization solution in the conventional SDVD plate can reach different concentration levels (in undersaturation, metastable, nucleation, and precipitation zones). Fig. 7 shows the schematics of the phase maps. In Fig. 7 a, for the SDVD plate, if the maximum concentration stays only in the undersaturation zone, the concentration level will not be enough for nucleation; hence, we will not be able to obtain any crystals in this case. For the two new plates, the situations are different. There are four initial concentrations for each plate, and there will be four maximum concentrations which are schematically shown in Fig. 7 a for each plate. Similar to the SDVD plate, when the initial concentration level is lower (volume ratios at 0.5 : 1.0, and 1.0 : 1.0), the maximum concentration may be still below the nucleation zone; therefore, no crystals will be formed. However, when the initial concentration level is higher (volume ratios at 1.5 : 1.0, and 2.0 : 1.0), the maximum concentration level may reach the nucleation zone or precipitation zone, i.e., the solution may reach or pass through the nucleation zone, and thus crystallization becomes possible. All of the other three cases (Figs. 7 b, c, and d) also show similar scenarios, i.e., the new plates can provide more opportunities for the crystallization solution to reach or pass through the nucleation zone, so that the probability of obtaining crystals is higher. The screening space of concentration using the two new types of plates is larger than using the conventional SDVD plate. In other words, the screening space of concentration is effectively expanded using the new plates. Due to cross diffusion in the CDM plate, the actual crystallization conditions found by the CDM plates are not completely the same as those found using the M plate. Therefore, although the total number of crystallization conditions found by both the CDM and the M plates is comparable, the


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crystallization conditions found by these two plates are complementary. Therefore, the combination of the two plates showed a significant increase in the number of crystallization screening hits. There are other techniques that explore the phase diagram, like free-interface diffusion (FD) in microfluidic systems, or counter-diffusion (CD) technique in capillaries. The concentration variation of FD or CD follows different paths

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in the phase diagram as compared with using

CDM or M plates. Apparently, utilization of FD or CD and utilization of CDM or M shall be complementary to each other. Combining both strategies can result in increased chances to obtain crystals. If the same initial concentrations (before mixing) for protein and precipitant are used for the different techniques, higher supersaturation level can be achieved by using CDM or M plates because these two plates allow continuous concentration. Furthermore, each CDM or M plate provides four different paths in the phase diagram simultaneously, hence larger region in the phase diagram can be explored than using FD or CD techniques. In the end of the discussions, it is also necessary to point out the imperfections of the new design as compared with the traditional SDVD method. To create multiple initial concentrations, the new design enables four crystallization droplets to be set up in one screening trial. Hence the consumption of protein sample may increase. However, more screening hits can be expected within shorter time, and furthermore, starting from lower initial concentrations will reduce the consumption of protein. Another issue necessary to address is that the concentration of the droplets in the new plates will continuously increase and eventually all droplets will dry out if the crystallization plates are not preserved in a humid environment. However, this issue is not a big problem because one can check the crystallization results frequently or use automation


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system to monitor the successful crystallization conditions, so as to provide valuable information for subsequent optimization process.


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Figure 7. Schematics of concentration evolution paths in the phase diagram during crystallization using different plates. Solid line, dash line, short dash line and dot line represent the concentration evolution paths for the ratios of protein and precipitant at 2.0 : 1.0, 1.5 : 1.5, 1.0 : 1.0 and 0.5 : 1.0, respectively. Solid circles (●), solid squares (■), and solid triangles (▲) represent the maximum concentration levels the solution can reach when the SDVD, CDM, and M plates, respectively, are used. Open diamond (◇) represents the initial concentration point. (a), (b), (c) and (d) show different cases when the maximum concentration levels for SDVD plates are in the undersaturation, metastable, nucleation, and precipitation zones, respectively. The phase diagrams illustrate that the crystallization solution in both CDM and M plates can reach higher supersaturation conditions, and wider concentration ranges, thus providing better chances for crystallization.


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Concluding remarks We propose in this paper a new design for protein crystallization plates to be used with the crossdiffusion microbatch and microbatch methods, and reported for the first time the comparison among three format plates – CDM, M and SDVD plates in crystallization screening hits. The new design of crystallization plates is different from the commercial ones in that: 1) The plate design (including M and CDM plates) is different from the commercial ones. There are indeed multiwell plates available commercially, like CombiClover™ Plate provided by Emerald Bio, CrystalQuick™ provided by Greiner Bio-One, and 3 Well Crystallization Plate provided by SWISSCI. These plates are multiwell plates containing a reservoir well in each unit. The new design does not contain any reservoir well, hence it can not only reduce the consumption of reservoir solution, but can also realize gradual concentration via permeable plate, which is especially useful for proteins with high solubility to achieve maximum crystallization hits; 2) The concept is very simple but it allows four different initial volume ratios to be screened simultaneously, so that one can finish four (or even more) possible concentration ratios in one experiment, which is efficient and easy to perform;3) The equilibrium occurs among all droplets in CDM plate or among the four droplets in one unit in M plates, which is different from those in the conventional vapor diffusion or microbatch methods. Apparently, the new design ensures that the concentration screening space can be effectively expanded without a significant increase in labor, and the screening results in this study confirmed that the performance in crystallization condition finding can be significantly improved, and thus the new design and the new plates are robust and easy to apply in practical protein crystallization screening.


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AUTHOR INFORMATION Corresponding Author *Phone: +86-29-88460254. Fax: +81-29-88460254. E-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. D.C.Y. designed research; C.D., C.Y.Z., Y.Y.L., R.B.Z. and Q.D.C. performed research; C.D., C.Y.Z., Y.Y.L. and Q.D.C. analyzed data; and C.D., C.Y.Z., Y.Y.L. and R.B.Z. wrote the paper. The authors declare no conflict of interest.†C.D. and C.Y.Z. contributed equally. Funding Sources National Basic Research Program of China (973 Program, Grant No. 2011CB710905) National Natural Science Foundation of China (Grant No. 11202167 and Grant No. 31170816) China Postdoctoral Science Foundation (Grant No. 2013T60890) Fundamental Research Foundation (FRF, 3102014JKY15006) of NPU in China Undergraduate Training Programs for Innovation and Entrepreneurship in China (Grant No. 201410699087) Notes The authors declare no completing financial interest.


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Acknowledgments This work was supported by the National Basic Research Program of China (973 Program, Grant No. 2011CB710905), the National Natural Science Foundation of China (Grant No. 11202167 and Grant No. 31170816), the China Postdoctoral Science Foundation (Grant No. 2013T60890), the Fundamental Research Foundation (FRF, 3102014JKY15006) of NPU in China, and the Undergraduate Training Programs for Innovation and Entrepreneurship in China (Grant No. 201410699087).


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ABBREVIATIONS CDM, cross diffusion microbatch; SDVD, sitting drop vapor diffusion; CDM plate, CDM plate 96-4; M plate, microbatch plate 96-4.


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For Table of Contents Use Only A new design of protein crystallization plates to expand concentration screening space in crossdiffusion microbatch and microbatch methods

Chen Dong, Chen-Yan Zhang, Yang-Yang Liu, Ren-Bin Zhou, Qing-Di Cheng, Da-Chuan Yin

Synopsis: A new design of protein crystallization plates aims to enable multiple initial concentrations is proposed for cross-diffusion microbatch and microbatch methods to expand the concentration screening space. The new method can dramatically increase the probability to obtain protein crystals hence it is potentially useful in practical protein crystallization.


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Figure 1. Crystallization plates used in this study. (a) CDM plate. It contains 96 units, and each unit consists of four wells. All 96×4 wells are in one common space so that any evaporated species can diffuse to any other droplets. (b) M plate. It contains 96 units, and each unit contains four wells. The units are separated but the four wells in one unit share the same common space. (c) SDVD plate. It contains 96 units. Each unit consists of two sitting-drop wells and one reservoir well.



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Figure 2. Number of crystallization hits at different volume ratios of protein and precipitant solutions. (a1) The number of crystallization screening hits using the CDM plate. (a2) The averaged normalized screening hits at different volume ratios. **P＜0.01, *P＜0.5. error bar: mean ± SEM, n =12; (b1) The number of crystallization screening hits using the M plate; (b2) The averaged normalized screening hits at different volume ratios. *P＜0.5. error bar: mean ± SEM, n =12. SEM: standard error of the mean. n: number of sample. Value of P represent reliability of result, *P＜0.5 indicates the difference are significant, **P＜0.01 indicates the difference are highly significant. 108x70mm (300 x 300 DPI)


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Figure 3. Examples of redundancy in crystallization screening using CDM and M plates. The droplet images in this figure show the crystallization results of lysozyme in unit A8 using a CDM plate, lysozyme in unit G11 using an M plate, proteinase K in unit A12 using a CDM plate, and proteinase K in unit B3 using an M plate at different volume ratios of protein solution to precipitant solution. It can be seen that, in the same unit, there might be more than one droplet that yields crystals. The redundancy was removed by counting the number of units that yielded crystals, i.e., any wells that yielded crystals in the same one unit count as one hit.



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Figure 4. Comparisons of the number of crystallization screening hits using different crystallization plates (SDVD Plate, M Plate, and CDM Plate). The number of crystallization screening hits for the M and CDM plates is without redundancy, i.e., hits in one unit count as one hit. (a1) Comparisons of the number of screening hits between CDM and SDVD Plates. (a2) The averaged normalized screening hits on the CDM plate compared to the SDVD Plate, **P＜0.01, error bar: mean ± SEM, n =12. (b1) Comparison of the number of screening hits between M and SDVD Plates. (b2) The averaged normalized screening hits on the M plate compared to the SDVD Plate, **P＜0.01, error bar: mean ± SEM, n =12. (c1) Comparison of the number of screening hits between CDM and M plates. (c2) The averaged normalized screening hits on the CDM plate compared to the M Plate. error bar: mean ± SEM, n =12. 154x140mm (300 x 300 DPI)


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Figure 5. Comparison of the number of crystallization conditions found by only one plate. (a) Comparison of the number of crystallization conditions found only by CDM plate and SDVD plate. (b) Comparison of the number of crystallization conditions found only by M plate and SDVD plate. *P＜0.05, **P＜0.01. Error bar: mean ± SEM. n=12. 59x21mm (600 x 600 DPI)



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Figure 6. Comparison of the number of crystallization screening hits using the combination of the two new plates versus the SDVD plate. (a) Number of screening hits using the combination of the two new plates (CDM and M plates), and using the SDVD plate. (b) Comparison of the averaged normalized crystallization screening hits using the combination and the SDVD plate. **P＜0.01. Error bar: mean ± SEM. n=12. 63x23mm (600 x 600 DPI)


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Figure 7. Schematics of concentration evolution paths in the phase diagram during crystallization using different plates. Solid line, dash line, short dash line and dot line represent the concentration evolution paths for the ratios of protein and precipitant at 2.0 : 1.0, 1.5 : 1.5, 1.0 : 1.0 and 0.5 : 1.0, respectively. Solid circles (●), solid squares (■), and solid triangles (▲) represent the maximum concentration levels the solution can reach when the SDVD, CDM, and M plates, respectively, are used. Open diamond (◇) represents the initial concentration point. (a), (b), (c) and (d) show different cases when the maximum concentration levels for SDVD plates are in the undersaturation, metastable, nucleation, and precipitation zones, respectively. The phase diagrams illustrate that the crystallization solution in both CDM and M plates can reach higher supersaturation conditions, and wider concentration ranges, thus providing better chances for crystallization.


A New Design of Protein Crystallization Plates To ... - ACS Publications

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