Efficient Screening Methodology for Protein Crystallization Based on

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An efficient screening methodology for protein crystallization based on the counter-diffusion technique Luis A. Gonzalez-Ramirez, Carlos R. Ruiz-Martínez, Rafael A. Estremera-Andújar, Carlos A. NievesMarrero, Alfonso Garcia-Caballero, Jose A. Gavira, Juan López-Garriga, and Juan M. García-Ruiz Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b01353 • Publication Date (Web): 31 Oct 2017 Downloaded from http://pubs.acs.org on November 2, 2017

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Crystal Growth & Design

An efficient screening methodology for protein crystallization based on the counter-diffusion technique Luis A. González-Ramírez1, Carlos R. Ruiz-Martínez2†, Rafael A. Estremera-Andújar3, Carlos A. Nieves-Marrero2, Alfonso García-Caballero1, José A. Gavira1*, Juan López-Garriga3 and Juan M. García-Ruiz1* 1

Laboratorio de Estudios Cristalográficos, Instituto Andaluz de Ciencias de la Tierra (CSICUGR), Avenida de las Palmeras 4, Armilla, Granada 18100, Spain. 2

Natural Sciences Department, University of Puerto Rico, Aguadilla Campus, P.O. Box 6150, Aguadilla PR, 00604-6150, Puerto Rico. 3

Chemistry Department, University of Puerto Rico, Mayaguez Campus, P.O. Box 9019, Mayagüez, P.R. 00681, Puerto Rico. *Correspondence email: [email protected], [email protected] †

This article is dedicated to Dr. Carlos Ruiz-Martinez who passed away on May 16, 2017 at the early age of 42.

Abstract We report an efficient screening methodology based on the capillary counter-diffusion technique (CCD), which was evaluated using two different practical approaches. The first consisted of kits prepared with the most successful crystallizing agents (PEG and ammonium sulphate) buffered at different pHs ranging from 4 to 9 and tested on 14 samples, including commercial and research target proteins. The second approach was based on the previously identified and highly effective 24 crystallization cocktails adapted to the counter-diffusion setup. This screening was tested with two target proteins, HbII and HbII-III from the clam Lucina pectinate, and the results compared with those obtained with the vapour-diffusion experiment. The success rate was higher than 60% in both approaches. These results experimentally confirm the usefulness of the CCD technique for the screening of crystallization conditions of biomacromolecules beyond its wellknown value for the growth of large and high-quality crystals. We describe a detailed protocol for the laboratory implementation of the capillary counter-diffusion technique. Keywords: Counter-diffusion; Crystallization; Screening; Capillary; Biological macromolecules; Proteins.

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1. Introduction The screening of crystallization conditions is widely acknowledged to represent the rate-limiting step to produce diffraction-quality crystals for structural proteomics and genomics.1-3 This is despite the rapid development of high throughput methods, which currently allow the miniaturization and automation of experiments,4 but which have not led to high output of crystallized proteins.1, 5 The success rate in going from cloned gene to high-resolution protein structure is still disappointingly low – a problem that is especially critical in going from purified protein to diffracting crystal.6 The most difficult step of the crystallization process is to find the right chemical conditions for inducing nucleation for crystal growth3, 7, so using a straightforward efficient screening methodology is essential to increase the crystallization success rate.8 Ideally, a good screening methodology should meet the following criteria: a) flexibility: it must adapt to the wide range of physical-chemical conditions of protein solutions; b) time-efficiency: it should be capable of producing crystallization hits in reasonable times; c) low protein consumption; d) easy handling: it should allow easy manipulation of the protein crystal; e) robustness to external environmental stress: it should facilitate easy transportation/transference from one environment to another. At present, the main limitation in finding an efficient screening methodology lies in the fact that the chemical cocktail (type of solvent, buffer, precipitating agent, concentration of protein and precipitating agent, pH value, temperature, etc.) required to successfully crystallize a large number of proteins is quite diverse, while there is usually a limited amount of biomaterial available.9 Crystallization screening of proteins and other macromolecular compounds are currently performed either by the vapour-diffusion or the microbatch techniques, vapour diffusion being the most commonly used. One of the main drawbacks of both methodologies is that, in spite of the enormous number of chemical conditions that are assayed (it may run to several thousand), the screening of supersaturation values is neglected. The typical protocol consists of trying a large number of chemical conditions, which are only capable of exploring a very short range of supersaturation conditions in a single experiment. This is the main reason why screening is currently performed with robotic systems capable of handling micro- or even nano-volumes.4, 10, 11 In fact, with the advent of proteomics, high throughput crystallization robotic systems have been developed to set up thousands of crystallization experiments using the sitting-drop configuration of the vapour-diffusion technique.12 Nonetheless, although protein volumes have been reduced to the hundreds of nanolitres, the consumption of protein is still significant due to the high number of crystallization cocktails that need to be assayed in order to screen different supersaturation values.13, 14 To address the above-mentioned drawbacks, the counter-diffusion technique offers several advantages that make it an excellent choice for screening purposes. The basis of the counterdiffusion screening lies in the fact that each of the components of the precipitating cocktail has a different diffusion coefficient giving way to the overlapping of four different gradients taking place concomitantly: the buffer molecules diffuse faster and change the pH of the protein solution, salt molecules diffuse almost simultaneously, then low molecular weight PEGs and finally high molecular PEG molecules scan the capillary. As a result, a wide range of conditions are tested in each counter-diffusion screening experiment.15, 16 This means that counter diffusion,


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unlike batch and vapour-diffusion methods, is capable of screening in one single experiment, for any given protein sample and precipitating agent or chemical cocktail, a wide range of supersaturation values and supersaturation rates. Although many target proteins have been crystallized by counter diffusion, to date there has only been one study that demonstrates the efficacy of counter diffusion for screening purposes, using three model proteins, lysozyme, thaumatin and insulin.17 A simple and rational approach for overcoming the large number of possible cocktail combinations is the development of screening kits based on the most successful conditions.18 While the number of crystallization kits offered on the market keeps increasing,13, 19-21 parallel studies 8, 12, 14, 22, 23have been carried out to demonstrate that there is no direct correlation between the number of different crystallization conditions assayed and the number of positive hits obtained. For instance, Kimber and co-workers23 screened 755 proteins and demonstrated that 94% of the total number of hits can be obtained using just 24 conditions. In this work we have used ad hoc crystallization screening kits based on the most successful precipitating agents typically employed in protein crystallization, namely three different polyethylene glycols ranging from low to high molecular weight (PEG 400, PEG 4000 and PEG 8000), plus a kit of ammonium sulphate, all of them at six different pH values, to test their efficiency against 14 different proteins. Additionally, we analysed whether the use of a mixture of the three PEGs as precipitating agent is as effective as the individual ones. Furthermore, we have adapted the 24 crystallization conditions suggested by Kimber and co-workers23 to the capillary counter-diffusion technique in order to maximize the screening. We present the results obtained with two target haemoproteins, HbII and HbII-III, from the clam Lucina pectinate, highlighting the benefits when compared with traditional crystallization techniques. 2. Materials and Methods Commercial proteins; trypsin, xylanase and apoferritin purchased from Sigma-Aldrich, and glucose isomerase (Gluci) purchased from Hampton Research, were used without further purification, while target proteins, donated by collaborating research laboratories, were used as supplied. Non-commercial samples were: wild type SH3 (Src Homology 3) domain (SH3-WT), anti-lysozyme camel antibody complexed with lysozyme (cAb-Lys3), lumazine, dehydroquinase (DHQ), dihydropirimidinase from Sinorhizobium meliloti (Ser38), triose-phosphate isomerase (TIM), SM-like protein (AF-SM1) from Archaeoglobus fulgidus (AFSM), Satellite Tobacco Mosaic Virus (STMV), pike parvalbumin (PikePA) and haemoglobin. The final protein concentrations ranged from 6 mg/ml to 34 mg/ml depending on previous knowledge or availability. All other reagents were of analytical grade purchased from Sigma Aldrich (St. Louis, USA). Solutions were prepared with ultrapure water (Milli-Q® Integral-3© Merck-Millipore). The Granada Crystallization Boxes-Domino® (GCB-D) and the Counter-diffusion Screening kit (GCB-CSK®) were purchased from Triana Science & Technology. 2.1. Haemoglobin preparation The haemoproteins, HbII and HbII-III, from the clam L. pectinata, were collected from a sulphide-rich mangrove ecosystem on the Caribbean Sea coast. This mollusc contains three haemoproteins: the sulphide reactive haemoglobin I (HbI) and two oxygen reactive



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haemoglobins, called haemoglobin II (HbII) and haemoglobin III (HbIII). HbII and HbIII can be obtained from the isolation of the native HbII-III oxygen carrier complex. The isolation and purification of all Hb’s were carried out in four steps, using previously established protocols.24, 25 Firstly, the HbII-III complex was isolated from raw extract using a Fast Protein Liquid Chromatography system (FPLC) with Size Exclusion Chromatography (SEC, HiLoad 26/60 Superdex 200 grade from ÄKTA, Amersham Bioscience) using 0.5 mM EDTA and 50 mM NaH2PO4 at pH 7.5 as buffer. Secondly, Ion Exchange Chromatography (IEC) was used to separate the HbII and HbII-III protein complexes employing NaCl salt gradient (buffer A: 10 mM sodium acetate and 10mM triethanolamine at pH 8.3; buffer B: 10 mM sodium acetate, 180 mM sodium chloride and 10 mM triethanolamine at pH 8.3), using a HiPrep 16/10 QFF column coupled with a HiTrap QFF (5 mL) pre-column, both from GE Healthcare. Thirdly, the oxyHbII and oxyHbII-III complexes were obtained after bubbling oxygen at 1 atm and characterized spectroscopically.24 Finally, the buffer at the end of the purification step was replaced by deionized water by centrifugation using concentrator cells (Amicon® Model 8050 with a Millipore® membrane YM10). The final concentration was monitored using UV-Vis spectroscopy.24 2.2. Preparation of polyethylene glycol (PEG) and Ammonium sulphate kits at pH 4 to 9. We used the GCB-D boxes to set up the kits.26 This device (70 x 7 x 17 mm) is made of polystyrene and has flat parallel surfaces allowing easy optical inspection of each capillary. The GCB-D boxes can hold more than six capillaries of the same protein or of different proteins. The crystallization kits tested in this work were made of three different polyethylene glycols (PEG) from low to high molecular weight (30% PEG 400, 30% PEG 4000, 30% PEG 800), a mix of them (20% PEG 400, 15% PEG 4000 and 10% PEG 8000) and ammonium sulphate (AS) at 3.0 M concentration. Each kit screening was prepared at six pH values using sodium acetate/acetic acid 0.1 M for pH 4.0 and 5.0, Bis-tris/HCl for pH 6.0 and Tris/HCl for pH 7.0, 8.0 and 9.0, at a final concentration of 0.1 M. Each GCB-D box was filled with 2.5 ml of each buffered crystallizing solution. Then, a 0.5% (w/v) agarose layer was slowly poured on top of the crystallizing solution and left to gel. Pre-cooling the boxes containing crystallizing solution at 4ºC may help the formation of the gel layer. The filling protocol is schematized in Figure 1 and the final kit composition summarized in Table S1. 2.3 Crystallization experiments using home-made precipitant solutions Crystallization experiments using the Capillary Counter-Diffusion (CCD) with PEGs and AS were set up using four commercial proteins (trypsin, glucose isomerase, xylanase, apoferritin), and ten target proteins (SH3-WT, cAb-Lys3, lumazine, DHQ, Ser38, TIM, AFSM, STMV, pike parvalbumin and haemoglobin). Each capillary (0.1 mm inner diameter and 40 mm long) was filled with protein solution by capillarity forces and the top-end sealed with plasticine. Each capillary was inserted through its open end into each pre-filled GCB-D box through the on-top agarose layer. Finally, the GCB-D boxes were closed with their caps and sealed with parafilm. Up to 5 capillaries were inserted per box. In total, 9 µL of each protein were used to set up the 30 experiments. The experiments were monitored after 24, 48, and 72 hours, then each week until one month (1st observation) and finally one year after (2nd observation). The effect of the different kits was


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analysed in terms of the number of conditions that produced crystalline material, “hits”, and also the time lapse to observing the first crystal. 2.4 Crystallization experiments using commercial crystallization screens In the second approach, we have studied the crystallization behaviour of two target proteins, HbII and HbII-III, using the 24 conditions of the Counter-diffusion Screening kit GCB-CSK® (Triana S&T). Capillaries of 0.2 mm inner diameter and 40 mm length were filled with protein solution to approximately 35 mm (1.1 µL) and the top-end sealed using plasticine. Each capillary was inserted into each pre-filled GCB-D box through the top agarose layer. Finally, the GCB-D boxes were closed with a rubber cap and sealed with parafilm.27 In total, 26.4 µL of protein solution were used for the full screen (24 conditions). Hanging-drop vapour-diffusion experiments (HDVD) were set up in parallel using 24 well VDX crystallization plates (Hampton Research) and the Hampton Research Screen I (50 conditions). Hanging droplets were prepared by mixing 2 µL of protein solution, HbII and HbII-III, with 2 µL of each reservoir solution on a 22-mm siliconized round-coverslip, inverted over a 1-mL reservoir. In total, 100 µL of each protein solution was used. Protein stock solution of HbII and HbII-III was prepared at 30mg/mL in ultrapure water. Experiments were incubated at 20°C and monitored after 1h, 24h, 48h and 72h; then each week until week 11 and finally at weeks 15, 18, 24 and 31. The crystals were easily identified because of their red colour. 3. Results and Discussion When considering the development of crystallization screening kits to be used with the capillary counter-diffusion (CCD) technique, we envisaged exploring two parallel routes. On the one hand, we aimed to explore protein surface charge by changing the pH from 4.0 to 9.0 while keeping constant the two more successful precipitating agents, i.e. ammonium sulphate and poly-ethylene glycol (PEG).1 Since PEG is available in a wide range of molecular weights we selected 400, 4K and 8K as the most representative. Considering that mass transport in the capillaries is mainly controlled by diffusion, we also included a cocktail mixture of the three PEGs in order to compare its efficiency against each individual PEG. This first group was therefore composed of five kits (AS, PEG400, PEG4K, PEG8K and a mixture of PEG400, 4K and 8K), and each kit is composed of 6 GCB boxes in which the pH varies from 4 to 9 in increments of one unit. On the other hand, it is well accepted that a short number of the spare matrix random screening conditions is sufficient to find suitable crystallization conditions in 94% of cases.23 Based on Kimber and co-workers’ analysis, our second approach was to adapt the 24 most successful conditions identified in their study to the counter-diffusion technique (see supplementary material Table S2). The same procedure can be applied to explore any other crystallization condition using a very simple set-up of the capillary counter-diffusion technique, the gel acupuncture method (GAME)16, 26, 28, (Figure S1) while keeping the same protein concentration. One of the most important advantages of the CCD crystallization screening is its low protein consumption. Employing the 5 kits at 6 different pHs and using capillaries of 0.1 mm inner diameter and 40 mm length, only 9 µL of protein solution are required for the full 30 screening experiments. For the second study, we employed capillaries of 0.2 mm requiring 26.4 µL of protein solution for the 24 conditions. If compared with the amount of protein required to



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manually set up a typical HDVD (hanging-drop vapour diffusion) full screening (approximately 100 µL), the use of CCD represents approximately a 4-fold reduction. Additionally, it is possible to use capillaries of 0.1 mm of inner diameter and 35 mm in length to reduce even further the volume of protein employed to just 0.24µl per capillary for a total volume of 5.7µL with the 24conditions screening kit. This amount is comparable to the amount of protein used for highthroughput robots (5.0µl of protein, 0.1µL per drop x 50 conditions). 3.1 Crystallization of proteins using home-made PEG and AS kits We followed the crystallization behaviour of fourteen different proteins ranging from low (7.2 kDa) to high (1000 kDa) molecular weight and covering the whole range of isoelectric points from 4.1 to 10.5. The five screening kits have pH values in the range from 4 to 9, imposing different net charges of the protein in solution, thus allowing us to reach some general conclusions. Table 1 summarizes the results of the crystallization screening using the four kits based on PEG and the ammonium sulphate. All fourteen proteins produced crystals in at least one of the 30 screening conditions. Figure 2 and S2 show examples of crystals obtained under different conditions. Using ammonium sulphate, 9 out of the 14 proteins (64%) produced crystals across the whole range of pH screened (Table S3). In three cases, cAb-Lys3, trypsin and AFSM, capillaries remained clear, indicating that supersaturation was not high enough. Interestingly in the case of apoferritin, which typically crystallizes in the presence of cadmium sulphate29, ammonium sulphate did not induce its crystallization, although the protein precipitated at pH close to the pI. This observed behaviour, from precipitation to clear capillaries or viceversa, was also observed with xylanase, and may be correlated with the lack of a necessary additive.30 Although in the case of xylanase we have not identified the missing additive, a fast check in the PDB (Protein Data Bank) shows that the majority of xylanase structures have been obtained with ligands of a different nature. It has also been observed that at values close to the protein pI, precipitation tends to be high and in the form of amorphous material or flocculates, whilst the formation of single crystals occurred at pH values far from the pI, with protein molecules charged either positively or negatively. These observations correlate well with the screening effect of salt anion and cations being more effective when the protein has a net charge.7, 9, 31 When the proteins were subjected to crystallization using the three individual PEGs or the mixture of PEGs, 13 out of the 14 proteins (93%) produced crystals (Table S4). Only DHQ shows the two states (precipitate/clear capillary), evidencing the lack of some relevant additive(s). It is worth mentioning that the three proteins that remained undersaturated with ammonium sulphate concentration of 2.8 M, produced crystals in the presence of PEGs. Furthermore, apoferritin and xylanase crystallized by the exclusion volume effect32 of high molecular weight PEGs, demonstrating that PEG is very efficient at increasing the supersaturation of any protein solution.33 This is why PEG is the most effective precipitating agent.34, 35 All the proteins that crystallized with any of the individual PEGs also crystallized with the combination of PEGs, except for cAb-Lys3, which only crystallized with PEGs 4K and 8K. From our results, it also seems clear that the mixture of PEGs has an additional affect, which drives the system to very high supersaturation values, thus yielding only precipitate. Therefore, the mixture of PEGs might be a very good precipitating cocktail to be used with less concentrated protein solutions.


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The capillary counter-diffusion technique allows fast contact between the crystallizing solution and the protein solution while maintaining the typical kinetics pattern inherent to the counterdiffusion technique, which spans the supersaturation values and supersaturation rates in a longer period of time, i.e. position along the capillary.15, 16, 36 This mass transport kinetics correlates well with the output of the experiments. Thus, when the system is sufficiently supersaturated, precipitation and crystals can be observed at the bottom of the capillaries within the first few days but the system does not reach equilibrium after one month for high molecular weight PEGs (1st observation, Table S5). In five of the cases (PikePA, Gluci, AFSM, xylanase and Tripsin), crystals were observed after the second observation (one year, Table S6), three of them only in the presence of one of the PEGs and two of them in both, PEG and in AS (Table 1). Crystals identified during the first observation were still in good condition from the optical point of view. 3.2 Crystallization of haemoglobins using commercial screening kits To test the efficiency of the CCD as screening technique, we selected two target proteins, HbII and HbII-III from L. pectinate. Since the main goal was to minimize the number of assays and protein consumption, a minimum screening of 24 conditions was designed based on the work of Kimber and co-workers.23 The 24 conditions suggested by Kimber have been adapted to the counter-diffusion set-up by increasing the concentration of the precipitating agents to the maximum of their solubility at 4ºC within the constraints of the cocktail mixture and preparation protocol. Buffer concentration, pH and additives were kept constant in all cases (Table S2). Each cocktail is composed of: a buffer that keeps the solution within one unit of pH (between 4 and 9); a salt to precipitate proteins by charge screening; a low molecular weight polyethylene glycol (PEG) and a high molecular PEG. Since the precipitating agent concentration is not diluted, as happens for vapour-diffusion experiments, crystallization experiments using the CCD technique start off far from equilibrium at high supersaturation values and so screen a broader range of conditions15-17. The evolution of the experiments was followed by optical microscopy at regular intervals over a period of 31 weeks (Table 2). A variety of crystal shapes and sizes were obtained, but only crystals that were obvious by their faceted shape and red colour or because of positive diffraction tests were considered as hits (Figure 2 and Figure S2). The CCD screen proved to be highly efficacious as it yielded crystals in 15 and 16 conditions for HbII and HbII-III, 63% (HbII) and 67% (HbII-III) of all the conditions tested, respectively. We have also compared this output with a similar experiment carried out by HDVD with the 50 conditions of the HR Screening I. The results have been summarized in Table S7 as a function of time. On the whole, the CCD based screen yields more crystallizing conditions than HDVD by the end of the screening experiment. In the case of HbII, the CCD screening produced 4 more hits (15) than HDVD (11), that is, a 36% increase. Furthermore, for HbII-III the CCD screening generated 4 more hits (16) than HDVD (12), that is, a 33% increment. However, it is more significant to evaluate the efficacy of each screening methodology by comparing the number of hits per number of conditions employed. From our results, the CCD screening yields crystals in 63% (HbII) and 67% (HbII-III) of all the conditions tested, whereas HDVD produces crystals in only 22% and 24% of the conditions for HbII and HbII-III, respectively. In other words, the CCD screening produces around 2.8 times more crystallization hits per condition employed.



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The crystallization cocktails of the CCD screening kit can be generally classified into four categories depending on the main precipitating agent (Table S8): (1) polyethylene glycols (PEGs), (2) organic or inorganic salts (Salts), (3) small organic molecules (SOM) and (4) mixtures of PEGs, SOM and salts in any combination or concentration (Mix).23 For the crystallization of HbII, the conditions of the CCD screening based on PEGs (62% success rate), salts (71% success rate) and their mixtures (100% success rate) were very successful while the two conditions including small organic molecules did not produce any crystal. For the crystallization of HbII-III, each type of condition showed very good results (around 50% success rate), especially those based on salts (100% success rate).37 We did not find a clear correlation between the molecular weight, i.e. diffusion rate, of the main crystallizing solution, and crystal nucleation time (meaning observation time). Chemical conditions that are based on small molecules such as salts and low molecular weight PEGs are very efficient at the beginning of the crystallization experiments due to their faster diffusion rate, while in the longer term only conditions containing large molecules such as PEGs are effective. A closer look at the hit rate shows that within the first hour of the screening, CCD produced 4 and 6 hits for HbII and HbII-III, respectively. Between the first hour and the third week, 10 and 9 new hits were observed, at a rate of 0.5 and 0.4 hits/day, for HbII and HbII-III, respectively. Importantly, after the third week both proteins still produced a new hit. Our observations reveal that CCD screening methodology produces relevant information at the early stages of the crystallization experiments, as expected from the higher initial supersaturation values, but also ensures a deep screening of the crystallization conditions along the capillary imposed by the different diffusion constant of the cocktail components. The use of CCD for screening purposes offers two additional advantages. Firstly, it is possible to acquire quality x-ray diffraction data directly from the capillary without further manipulation.38, 39 Secondly, CCD experiments show high mechanical stability, practically no evaporation and, most importantly, the crystals are protected against thermal changes and osmotic stress due to the high concentration of crystallizing agents. 4. Conclusions We have tested several crystallization screening kits based on the counter-diffusion technique (CCD) using two different approaches. The first consisted in using new kits designed ad hoc, taking into account the most successful precipitating agents (PEG and ammonium sulphate) combined with the modulation of the protein charge by changing the pH. In this case, the kits used were PEGs from low to high molecular weights (400, 4000 and 8000), a combination of them and the ammonium sulphate kit, prepared at 6 different pH values (4, 5, 6, 7, 8 and 9). It was observed that the use of the mixture of PEGs was more efficient than the use of AS and produced similar results to each individual PEG. The combination of the five screening kits (PEGs and AS) crystallized 100% of our test samples, but using just the mixture of PEGs and the AS kits (12 conditions) we were able to crystallize 13 out of 14 samples (94%). For the second approach, which is based on the 24 conditions of the minimal screen cocktails described by Kimber and co-workers23, the two target proteins, HbII and HbII-III, crystallized in 63% and 67% of the conditions, respectively. With these studies, it has been demonstrated that both approaches employed minimize the consumption of protein solution while maximizing the number of crystallization hits.


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Additionally, CCD has several other advantages: a) the wider range of crystallization conditions screened in each capillary yields crystals of high quality, which can be diffracted directly without further optimization; b) it is time-efficient, producing many hits within the first hour of the crystallization experiment as well as for long time periods of incubation; c) the robustness of the technique allows easy manipulation of the experiments and handling of the protein crystals; d) at the end of the crystallization each capillary holds the entire history of the experiment without having to record its evolution. Acknowledgments This work was partly supported by funds from the National Science Foundation, (NSF-MCB0843608) and the National Institute of Health (NIH-NIGMS-RISE-10421223). The Project "Factoría de Cristalización" CSD2006-00015, Consolider Ingenio-2010 (MICINN) and Proyecto de Excelencia RNM-5384 of the Junta de Andalucía provided financial support for this work. J.A.G acknowledges the financial support of project BIO2016-74875-P of the MICINN.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Experimental set-up of counter-diffusion experiments using the Gel Acupuncture Method, photographs of grown protein crystals, composition of PEG and ammonium sulphate crystallization screening kits, difference in composition between the 24-CCD and the corresponding 24 conditions of the 50-HDVD crystallization screening kits, crystallization outputs obtained when using ammonium sulphate at pH 4 to 9, crystallization results from polyethylene glycols of three different molecular weights and the mixture of them, crystallization results after one month and one year, crystallization conditions of the HDVD experiments crystals of HbII and HbII-III, and success rate of the CCD crystallization screening as a function of the main precipitating agent.

Table 1. Time lapse to observe the first crystals of each protein and the kits that produce the crystals.



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Protein PikePA Gluci AFSM SH3-WT Lumazine Apoferritin TIM DHQ Ser38 HbII cAb-Lys3 Xylanase STMV Trypsin

Conc. (mg/ml) 10 10 10 18 20 11 10 20 34 10 10 10 10 10

pI 4.1 4.8 5.0 5.3 5.4 5.5 5.6 5.9 5.9 6.5 8.4 9.0 10.0 10.5

MW (kDa) 11.4 173 60.2 7.2 1000 440 114 205 55 17.7 15.7 21 24

Time (Day) >30 >30 >30 4 3 5 3 4 1 4 3 >30 2 >30

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Kit AS, PEG 4K, PEG 8K, 3-PEG AS, PEG 400, PEG 4K, PEG 8K, 3-PEG PEG 4K, PEG 8K, 3-PEG AS, PEG 4K, PEG 8K, 3-PEG AS, PEG 4K, PEG 8K, 3-PEG PEG 4K, PEG 8K, 3-PEG AS AS AS, PEG 400, PEG 4K, PEG 8K, 3-PEG AS PEG 4K, PEG 8K 3-PEG AS, PEG 400, PEG 4K, PEG 8K, 3-PEG PEG 4K, PEG 8K, 3-PEG


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Table 2. Summary of crystallization conditions of the 24-CCD kit producing crystals of target proteins HbII and HbII-III at different times of the experiments (h = hours; d = days; w = weeks). Previous observed hits are highlighted in blue.

Protein HbII HbII-III HbII HbII-III HbII HbII-III HbII HbII-III HbII HbII-III

1 2 3 4 5 X X X X

X

X

X

X

X

6

Conditions of the CCD screening kit* Hits Time 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 X X X X 4 1h X X X X X 6 6 1d X 8 X X X X X 14 1w X X X 11 14 3w X X 15 X 15 31 w X 16



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Figure 1.

Glass capillary

Agarose gel layer Crystallizing agent GCB-Domino

1

Protein solution

4

3

2

Insertion of capillaries

Agarose gel layer

Crystals

Protein Solution Crystallizing agent

6 5 7 8 Figure 1. Scheme illustrating the experimental set-up of the counter-diffusion technique using the GCB-D box. Each GCB-D is filled with a crystallizing solution buffered at different pH values (4-9). After the crystallizing solution is poured into the GCB, a small amount of agarose solution is added on top and left to gel. This layer helps to keep the capillaries vertical. Then, each thin capillary (0.1 mm diameter) is filled with the protein solution by capillarity forces and one of its sides is sealed. The capillaries are inserted into the GCB and the box closed with its lid.


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Figure 2

Figure 2. Photographs showing representative crystals of SH3-WT in the 3-PEG kit at pH 9 (A); Trypsin in PEG 4000 at pH 9 (B); SMTV in PEG 8000 at pH 5 (C); Lumazine in PEG 4000 at pH 7 (D); HbIIIII (E); and HbII (F) obtained by the capillary counter-diffusion technique.



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References (1) Fazio, V. J.; Peat, T. S.; Newman, J., Crystallization: Digging into the Past to Learn Lessons for the Future. In Structural Proteomics: High-Throughput Methods, Owens, R. J., Ed. Springer New York: New York, NY, 2015; pp 141-156. (2) Luft, J. R.; Wolfley, J.; Jurisica, I.; Glasgow, J.; Fortier, S.; DeTitta, G. T., Macromolecular crystallization in a high throughput laboratory - the search phase. J. Cryst. Growth 2001, 232, 591-595. (3) McPherson, A., Macromolecular crystallization in the structural genomics era. Journal of Structural Biology 2003, 142, 1-2. (4) Shaw Stewart, P.; Mueller-Dieckmann, J., Automation in biological crystallization. Acta Crystallographica Section F 2014, 70, 686-696. (5) Bolanos-Garcia, V. M.; Chayen, N. E., New directions in conventional methods of protein crystallization. Progress in Biophysics and Molecular Biology 2009, 101, 3-12. (6) Rupp, B., Reviewing biomolecular crystallography proposals: time for a paradigm change. Trends in Biochemical Sciences 2015, 40, 419-421. (7) McPherson, A.; Gavira, J. A., Introduction to protein crystallization. Acta Crystallographica Section F 2014, 70, 2-20. (8) Luft, J. R.; Newman, J.; Snell, E. H., Crystallization screening: the influence of history on current practice. Acta Crystallographica Section F 2014, 70, 835-853. (9) Gavira, J. A., Current trends in protein crystallization. Archives of Biochemistry and Biophysics 2016, 602, 3-11. (10) Chayen, N. E., High-Throughput Protein Crystallization. In Advances in Protein Chemistry and Structural Biology, Andrzej, J., Ed. Academic Press: 2009; Vol. 77, pp 1-22. (11) Newman, J.; Pham, T. M.; Peat, T. S., Phoenito experiments: combining the strengths of commercial crystallization automation. Acta Crystallographica Section F 2008, 64, 991-996. (12) Rupp, B., Biomolecular Crystallography: Principles, Practice, and Application to Structural Biology. ed.; Garland Science: New York, 2009; p 800. (13) Luft, J. R.; Collins, R. J.; Fehrman, N. A.; Lauricella, A. M.; Veatch, C. K.; DeTitta, G. T., A deliberate approach to screening for initial crystallization conditions of biological macromolecules. Journal of Structural Biology 2003, 142, 170-179. (14) Newman, J.; Egan, D.; Walter, T. S.; Meged, R.; Berry, I.; Ben Jelloul, M.; Sussman, J. L.; Stuart, D. I.; Perrakis, A., Towards rationalization of crystallization screening for small- to medium-sized academic laboratories: the PACT/JCSG+ strategy. Acta Crystallographica Section D 2005, 61, 1426-1431. (15) Ng, J. D.; Gavira, J. A.; García-Ruíz, J. M., Protein crystallization by capillary counterdiffusion for applied crystallographic structure determination. Journal of Structural Biology 2003, 142, 218-231. (16) Otálora, F.; Gavira, J. A.; Ng, J. D.; García-Ruiz, J. M., Counterdiffusion methods applied to protein crystallization. Progress in Biophysics and Molecular Biology 2009, 101, 2637. (17) Ng, J. D.; Gavira, J. A.; Garcı́a-Ruı́z, J. M., Protein crystallization by capillary counterdiffusion for applied crystallographic structure determination. Journal of Structural Biology 2003, 142, 218-231. (18) Jancarik, J.; Kim, S.-H., Sparse matrix sampling: a screening method for crystallization of proteins. Journal of Applied Crystallography 1991, 24, 409-411.


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An efficient screening methodology for protein crystallization based on the counterdiffusion technique Luis A. González-Ramírez1, Carlos R. Ruiz-Martínez2†, Rafael A. Estremera-Andújar3, Carlos A. Nieves-Marrero2, Alfonso García-Caballero1, José A. Gavira1*, Juan López-Garriga3 and Juan M. García-Ruiz1*

TOC graphic

Synopsis The capillary counter-diffusion technique is a very simple way to search for initial crystallization conditions. Different set-ups and approaches are explained and their benefits highlighted using several model and target proteins.


Efficient Screening Methodology for Protein Crystallization Based on

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