Method for preparing high quality crystals by directing ionized gas molecules through and/or over a saturated solution comprising a protein

Abstract

Disclosed is a method for facilitating preparation of high quality crystals suitable for X-ray crystallographic studies. The method comprises that an electric charge or current is provided to a saturated solution of the molecule to be crystallized, preferably via a jet of gaseous ions. Also disclosed is an assembly for carrying out the method of the invention.

Claims

1. A method for preparing crystals of a substance comprising establishing an electric current through and/or over a saturated solution comprising a protein, said electric current being established by directing a jet, flux or stream of ionized gas molecules, which carries a net electric charge, towards said saturated solution, thereby facilitating crystal formation and/or crystal growth and improving diffraction quality of the crystals in said saturated solution, where improved diffraction quality is measured in terms of higher signal intensity, higher resolution, lower mosaicity, and lower R.sub.merge.

2. The method according to claim 1, wherein the protein is selected from the group consisting of a monomer and multimer protein.

3. The method according to claim 1, wherein said saturated solution is supersaturated.

4. The method according to claim 1, wherein said saturated solution comprises a solvent selected from the group consisting of an organic, inorganic or supercritical solvent.

5. The method according to claim 1, wherein is established an electric current through and/or over said saturated solution.

6. The method according to claim 1, wherein the jet, flux or stream of gas ions is provided by a gas ion transmitting device, which is optionally connected to said saturated solution via a return electrode, thereby establishing an electric circuit.

7. The method according to claim 5, wherein the electric current is at least 0.1 A and at most 100 A.

8. The method according to claim 1, wherein the net electric charge or electric current or electric field is provided until at least nucleation of crystals is expected to occur or until crystals are observed in said solution.

9. The method according to claim 1, wherein the net electric charge or electric current or electric field is provided intermittently or constantly.

10. The method according to claim 9, wherein a constant or intermittent DC current is applied or wherein a constant or intermittent AC current is applied.

11. The method according to claim 1, wherein said ionized gas molecules are ions of molecules from atmospheric air.

12. The method according to claim 1, wherein said electric charge or current is carried by negatively charged gas ions.

13. The method according to claim 12, wherein the negatively charged gas ions are O.sub.2.sup. ions.

14. The method according to claim 1, wherein said electric charge or current is carried by positively charged gas ions.

15. The method according to claim 14, wherein the positively charged gas ions are N.sub.2.sup.+ ions.

16. The method according to claim 1, wherein the jet, flux or stream of ionized gas molecules is projected directly onto said saturated solution.

17. The method according to claim 1, wherein the jet, flux or stream of ionized gas molecules is applied for a period of time of at least 1 second.

Description

LEGENDS TO THE FIGURE

(1) FIG. 1: Phase diagram applying to crystal growth. The bold line (solubility curve) divides phase space into regions that support crystallization processes (supersaturation solutions) from those where crystals will dissolve (unsaturated solutions).

(2) FIG. 2: The process of formation of 2- and 3-dimensional nuclei. In A monomers are associated to form a 2-D island. Later (B and C) aggregates give rise to multilayered stacks.

(3) FIG. 3: Schemes illustrating crystallization methods. FIG. 3A shows the hanging drop crystallization method. B shows the sitting drop crystallization method.

(4) FIG. 4: Schematic drawings of practical implementations of the present invention.

(5) A: Implementation, where a discharge electrode has an area that exceeds the area of the wells in a microtiter system used for crystallization.

(6) B: Implementation, where a discharge electrode has an area that does not exceed the area of the wells in a microtiter system used for crystallization.

(7) FIG. 5: Photograph of a practical implementation of the present invention.

DETAILED DISCLOSURE OF THE INVENTION

Definitions

(8) Providing a net electric charge means that a sufficient amount of electric charge is provided to a site, whereby an electric current passes from said site to surrounding area for an appreciable period of time, or whereby an electrostatic charge is build up at said site. This means that a current has to be established for at least 1 second, but often longer, such as at least 10, 20, 30, 40, 50 or 60 seconds, or even at least several (2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30) minutes.

(9) Ionized gas molecules broadly refers to gas molecules, which carry a net electric charge. Typical examples are negatively charged oxygen molecules (O.sub.2.sup.) positively charged Nitrogen molecules (N.sub.2.sup.+) because of the abundancy of oxygen and nitrogen in atmospheric air, but any gas molecule capable of carrying a net charge is useful in the practice of the present invention. The important effect to achieve is simply that the jet, flux or stream provides the saturated composition with a net charge influx.

(10) In the present specification and claims a substance denotes a molecule or assembly of molecules (e.g. a complex) which is capable of forming crystals. It is generally believed that the method of the invention will facilitate production of crystals from all kinds of substances, but it is contemplated that the present invention will be particularly useful in providing crystals of macromolecular compounds and macromolecular complexes (e.g. when crystallising or co-crystallising molecules with a view to obtain X-ray crystallographic data).

(11) A macromolecular compound is a compound created by polymerization of smaller molecules and typically has a molecular weight in excess of 1 kDa. The term also embraces within its scope functional substances like multimeric proteins (which may be linked solely by non-covalent bonds and hence are normally considered to be multimers according to standard IUPAC terminology).

(12) A macromolecular complex is a non-covalently linked complex between at least one macromolecular compound and another substance. Typical examples of interest are antibody-antigen complexes, complexes between receptors and ligands and also complexes between enzymes and known binding partners (e.g. competitive antagonists for the normal substrate of the enzyme).

(13) A saturated solution is a solution where a substance is present in a maximum concentration under the ambient temperature and pressure, so that no further substance can be solubilised in said solution.

(14) A supersaturated solution is a saturated solution where more of the dissolved substance is present than could be dissolved by the solvent under the solubility amount. Supersaturated solutions are prepared or result when some condition of a saturated solution is changed, for example when increasing temperature, decreasing volume of the saturated liquid (as by evaporation), or by increasing pressure.

(15) The term nucleic acid denotes DNA and RNA, but also analogues thereof such as PNA and LNA.

(16) A gas ion transmitting device is a device or apparatus, which is capable of generating a jet, flux or stream of ionized gas molecules having a net electric charge and where the direction of the jet, flux or stream may optionally may be controlled with respect to dosage, intensity and direction. Specifics concerning such devices are discussed below.

EMBODIMENTS OF THE INVENTION

(17) As will appear from the examples, the present invention has been successfully used in the development of crystals under conventional conditions where crystals are not expected to appear and additionally has increased the incidence of crystallization, the total number of crystals that eventually appear and has improved the diffraction crystal quality.

(18) This underscores that treatment with ionized gas molecules provides an effective means for facilitating crystallization and involves several advantages, since it is easy and practical to administerfurther, the treatment of crystallisable solutions with ionized gas can in practice be combined with any means and method known in the art for preparing crystals. So, knowing from the prior art that ionic strength has been utilized successfully for the same scope, the present inventors have concluded that delivery of ionized gas molecules according to the teachings herein will be an advantageous improvement in other known technologies for crystal growth and crystallization.

(19) The first aspect of the invention relates to a method for preparing crystals of a substance, comprising

(20) a) providing a net electric charge to a saturated solution comprising said substance, thereby facilitating crystal formation and/or crystal growth in said saturated solution, or

(21) b) establishing an electric current through and/or over a saturated solution comprising said substance, said electric current being established by directing a jet, flux or stream of ionized gas molecules, which carries a net electric charge, towards said saturated solution, thereby facilitating crystal formation and/or crystal growth and improving the diffraction crystal quality in said saturated solution, or
c) establishing an electric field in which a saturated solution comprising said substance is located, thereby facilitating crystal formation and/or crystal growth and improving the diffraction crystal quality in said saturated solution.

(22) In embodiments of the first aspect of the invention, said substance is selected from the group consisting of a salt, an amino acid, a peptide, a protein, a carbohydrate, an amine, an alkane, an alkene, an alkyne, an aromatic compound, a heterocyclic compound, an alcohol, an organometallic compound, and a carboxylic acid. The invention has shown particular promise when crystallizing high MW molecules or complexes that notoriously may be difficult to crystallize, so it is preferred that the substance is a macromolecular compound, or complex. Typically, such a macromolecular compound is selected from the group consisting of a monomeric or multimeric protein; a nucleic acid; a polysaccharide; and a lipid, and typically the complex comprises a macromolecular compound, such as a complex of an antibody bound to an antigen or a receptor bound to a ligand.

(23) According to the invention, the (super)saturated solution typically comprises a solvent selected from the group consisting of an organic, inorganic or supercritical solvent.

(24) In the embodiments falling within option a) of the first aspect, that is, when the crystallization is facilitated by providing a net electric charge, the saturated solution should be electrically insulated from surrounding, so as to ensure that the charge provided will not discharge within a very short while. In these embodiments, the charge is typically delivered to said solution by means of a suitable device. For instance, a gas ion emitting device described in detail herein constitutes one advantageous possibility, but other means for delivering charge are believed to be equally useful. For instance, the group of devices known as electrostatic generators can be used; examples are a Van de Graaf generator, a Wimshurst machine, and a pellotron. Alternatively the device can be a friction machine which uses the triboelectric effect. One possibility is to supply charge from one of these devices where a container comprising the solution to be crystallized is connected to the terminal where charge is delivered. In such a setup, the delivered charge will become substantially evenly distributed over the combined area of the terminal of the electrostatic generator and of the container.

(25) When convenient the provision of electric charge is terminated or interrupted in the first aspect of the invention, which means that no more charge will be added. Over time, the solution will hence become electrically neutralized due to loss of charge to the surrounding environment (for instance due to interaction with atmospheric air), but this may take considerable time. Typically, the termination or interruption is therefore accompanied or followed by a step of actively neutralizing the polarity of said saturated solution before the crystals obtained are processed further. This may be achieved by directly or indirectly grounding said saturated solution (contacting the container with a ground electrode if the container is made of a conductive material or by contacting the saturated solution directly with a ground electrode. Alternatively, the saturated solution can be subjected to ionized gas molecules of opposite polarity of the ionized gas molecules used to provide said net electric charge, for instance by supplying a jet, flux or stream of gas molecules in a mixture comprising both positively and negatively charged ionised gas molecules.

(26) In interesting embodiments of the first aspect of the invention, option b) mentioned above is employed, that is, the crystallization is facilitated by establishing an electric current through and/or over said (super)saturated solution. This may according to the invention be achieved by providing the jet, flux or stream of gas ions from a gas ion transmitting device, which may in turn be connected to the (super)saturated solution via a discharge electrode (in this case a return electrode), thereby establishing an electric circuitthis enables a convenient way of controlling the administration of gas in response to the current that e.g. can be measured in the conductor between the return electrode and the ion transmitting device. Alternatively, the (super)saturated solution may simply be grounded (in this case the discharge electrode is not a return electrode)in such a setup, the current that is led to ground may be used as input to control gas ion dosage. In both circumstances, the normal range of operation utilises a current of at least 0.1 A and at most 100 A, with typical values of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.9, 1, 2, 3, 4, and 5 A.

(27) The ionized gas molecules (also termed air ions) are hence conveniently provided from a gas ion transmitting device, which includes a gas ion generator (also known as an air ionizer). This type of device is known to the skilled person and is e.g. used in the semiconductor industry to counteract electrostatic charge building when handling and transporting microchips and wafers. However, a suitable gas ion transmitting device for use in the present invention is one which, apart from generating the ions, is capable of delivering a directed stream of gas ions having either a predominant positive or a predominant negative charge (where e.g. the devices used in the semiconductor industry typically deliver a balanced mixture of positive and negative ions).

(28) In brief, such an apparatus generates a high voltage applied to an electrode, which thus produces an electric field that is most intense in a defined region (e.g. immediately adjacent a sharply pointed tip). The intense electric field disrupts the normal charge state of molecules of air gases (e.g. nitrogen and oxygen) in the region adjacent to the sharply pointed tip and some of the molecules become negative or positive ions, depending upon whether the molecule attains an excess or a deficiency of electrons (typically oxygen will become negatively charged, whereas nitrogen will become positively charged). The ions having a polarity opposite from the polarity of the high voltage of the electrode are attracted to the electrode and are neutralized, whereas ions of the same polarity as the high voltage electrode are repelled by the electrode and are dispersed outwardly. The subsequent projection towards the treated object with the thus generated air ions can be controlled when the receiving object is connected to a discharge electrode of opposite or neutral polarity (i.e. a ground electrode), which will ensure a correct projection of the ionized gas molecules in the desired direction towards the target area. Also, such a discharge electrode can be equipped with an amperemeter or other device for measuring the current passing through the electrode whereby the dosage of the ionized gas molecules can be controlled by a feed-back mechanism where gas ion generation and projection is controlled in response to the current passing through the discharge electrode (i.e. if the current exceeds a preselected current, the amount of gas ions projected by the ionized gas transmitting device is down-regulated, and vice versa).

(29) Known suitable devices are disclosed in WO 2007/042029.

(30) In embodiments of the first aspect of the invention, the net electric charge or electric current or electric field is provided until at least nucleation of crystals is expected to occur or until crystals are observed in said solution. However, longer durations of exposure may be advantageous in terms of crystal diffraction quality.

(31) The net electric charge or electric current or electric field may be provided intermittently or constantly, e.g. a constant or intermittent DC current or wherein is applied a constant or intermittent AC current.

(32) The nature of the ionized gas molecules are not believed to be critical, since it is their capability of carrying electric charge which is of highest relevance. However, it is practical that the ionized gas molecules when used in embodiments of the present invention are ions of molecules from atmospheric air. For instance they may be negatively charged gas ions, such as O.sub.2.sup. ions, or they may be positively charged gas ions such as N.sub.2.sup.+ ions.

(33) In convenient embodiments, the jet, flux or stream of ionized gas molecules is projected directly onto said saturated solution. As mentioned above, this is often done at least until nucleation is expected to occur or until crystal formation is observed, but it is also possible to operate with fixed intervals of time. Typically, the jet, flux or stream of ionized gas molecules is applied for a period of time of at least 1 second, but substantially longer time may be necessary or practicalat least 10, at least 30, or at least 50 seconds, at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, etc. In practice, the method may be carried out over several hours (at least 2, 3, 4, 5, 6, 8, 12, 15, 20) or even several (at least 2, 3, 4, 5, 6, 7, 8, 9 or 10) days.

(34) The second aspect of the invention relates to a method for preparing crystals of a substance, comprising establishing an electric current through and/or over said saturated solution thereby facilitating crystal formation and/or crystal growth and improving the diffraction crystal quality in said saturated solution. In this aspect, the substance as well as the (super)saturated solution have the same characteristics as discussed in respect of the first aspect of the invention. When not using ionized gas for delivery of the electric current, the electric current can be established via at least one cathode and at least one anode, which are each in contact with said saturated solution and which are connected to an electrical power source, which in embodiments is adapted to deliver an electric current of at least 0.1 A and at most 100 A. As is also the case with the first aspect of the invention, the electric current is in some embodiments provided until at least nucleation of crystals has been observed in said solution, and it is also possible to provide the electric current intermittently or constantly, such as in the form of a constant or intermittent DC current or a constant or intermittent AC current. Also, the time for applying the current may be as discussed in respect of the first aspect of the invention.

(35) In both the first and second aspects of the invention, crystallisation may further be facilitated by subjecting the saturated solution to any one of the following: cooling, heating, solvent evaporation, pressure change and solvent composition alteration.

(36) The third aspect of the invention relates to use of ionized gas molecules in the preparation of crystalsit is to be understood that such use can incorporate all the features discussed in the context of the first aspect of the present invention.

(37) Finally, the fourth aspect of the invention relates to an assembly suitable for preparing crystals of a substance, comprising a container that can accommodate at least one sample of a saturated or supersaturated solution of said substance, a gas ion transmitting device, which is adapted so as be capable of delivering a jet, flux or stream of ionised gas molecules that carries a net electric charge, and which is arranged relative to said container so that said jet, flux or stream of ionised gas molecules will be projected onto said saturated solution in said container, optionally an electrode, which is arranged relative to said containing so as to facilitate that said jet, flux or stream is projected towards with said saturated solution, and a conductor that connects said electrode to either said gas ion transmitting device or to ground, whereby an electric current can be carried via said ion transmitting device, said jet, flux or stream of ionized gas molecules, optionally said saturated solution, said electrode, and said conductor.

(38) In other words, this assembly of the invention embodies the gas ion delivery related method of the first aspect of the invention in a practical setup, and therefore all features that characterize this part of the first aspect of the invention (choices pertaining to the substance, the saturated solution, the ionized gas molecules, the timing and form of gas ion delivery etc) apply mutatis mutantis to the fourth aspect of the invention. Consequently, the assembly of the invention is in certain embodiments one, wherein said ion transmitting device includes a control for setting said electric current and/or setting the timing of the delivery of said jet, flux or stream and/or setting the phase of said electric current and it is preferred that this is done so as to carry out the method of the first aspect of the present invention. For instance, it is preferred that the ion transmitting device is capable of delivering a jet, flux or stream of O.sub.2.sup. and/or N.sub.2.sup.+.

(39) One practical embodiment is to have the control adapted so as to allow adjustment of said electric current in response to the electric current passing through the conductor. As mentioned above, this can result in two different modes of operation: if the saturated solution to be crystallized is insulated from the surroundings, the adjustment will serve to ensure a controlled projection of charged ions in the correct direction of the saturated solution thereby facilitating preservation of a stable electrostatic state, and if the saturated solution is in contact with the return electrode, the adjustment can directly control a gas ion generated current passing through or over the solution.

(40) The fifth aspect of the invention relates to an assembly (or apparatus) suitable for preparing crystals of a substance, comprising a container that can accommodate at least one sample of a saturated or supersaturated solution of said substance, said container being electrically insulated from its surroundings, an electrostatic generator or a friction machine, which is arranged so as to provide said container with a net electric charge. The latter may, as explained above, be arranged relative to the container is such a way that the generator/friction machine has a terminal which accumulates the charge delivered by the device and that said terminal is in contact with the container and solution so as to allow charge to distribute itself over the combined area of the terminal and the container/solution. Useful electrostatic generators are described above.

(41) The practical implementation of the inventive methods and assembly typically entails that a drop containing the molecules to be crystallized is exposed for a period of time to a jet, flux or stream of ionized gas molecules which carry a net charge sufficient to generate new crystallization nuclei or to increase the number of the already formed nuclei.

(42) One set up system for both investigation of the influence of wireless current on the crystals and for implementation of the method of the invention is schematically shown in FIGS. 4A and 4B. A container (e.g. an open multi-well (8-96) plate with sitting or hanging droplets) comprises the molecules to be crystallized is subjected to a jet, flux or stream of gas ions from a gas ion transmitting device (WMCS in the figure). The plate is connected to a discharge electrode situated under the container. A control system operates by adjusting the flow of ions in response to the current returned from the discharge electrode. In some embodiments, the discharge electrode is arranged so as to be electrically insulated relative to (super)saturated liquid in the platein those embodiments, the discharge electrode mainly serves as a means for accurately directing the gas ions towards the plate so that an electrostatic charge can be provided. In other embodiments, the discharge electrode is in direct contact with the liquid, thus enabling the establishment of a current in a closed circuit consisting of the ion emitting device, the gas ions, the solution to be crystallised, the electrode and a conductor from the electrode to the ion emitting device.

(43) FIG. 5 shows a picture of an experimental setup, where a gas ion emitting device is located on a table and a non-conducting polysterene multi-well plate is suspended and attached to a return electrode. In this setup, the gas ions emitted by the device will be directed to the multi-well plate and charged ions will be distributed over the surface. Thus in this setup, there is not established any current through or over the crystallisation solution present in the wellsrather, the continuous delivery of gas ions that are targeted towards the plate by the return electrode will cause a slight accumulation of charge on the plate surface and any loss of charge over time to the surroundings will be compensated by the delivery of gas ions.

(44) It is to be noted that the use of a discharge electrode is truly optional, albeit practical. If situating the gas ion emitting device optimally relative to the crystallisation solution and the plate or container comprising it, the delivery of gas ions will be sufficient to provide the necessary charge distribution.

(45) These setups allow investigation into the effect of the wireless current on the crystallization process of a macromolecule or macromolecular complex. In the Example referenced below, crystallization was investigated using a model protein, Hen Egg White Lysozyme (HEWL). Charge carrying ions were dispensed over the suspension for 4 and 8 minutes (plate 2 and plate 3 respectively) and the crystal formation was visually checked by a conventional optical microscope. After the exposure to the ions plates were stored at room temperature. The results show that the crystal production and the crystal quality increases with the time of wireless current treatment cf. the Example for more detail.

Example 1

Crystallization of a Model Protein, HEWL

(46) Hen egg white lysozyme (HEWL) was the second protein and the first enzyme ever studied by X-ray diffraction and is still the most widely used for crystal growth studies. HEWL is an enzyme which hydrolyzes polysaccharides in bacterial cell walls. It is composed of 129 amino acids and has a molecular weight, M.sub.R, of 14,296 Da (Da=dalton=1.6610-27 kg). Lysozyme is particularly attractive for crystal growth research, because detailed information exists on its thermophysical properties (contrary to the situation for many other proteins).

(47) Crystallization Conditions:

(48) A preparation of 75 mg/ml HEWL, in 0.1 M NaAc pH 4.8 was prepared. The precipitation buffer was constituted of 6.5% (w/v) NaCl and 0.1 M NaAc pH 4.8. The protein solution was diluted in 0.1 M NaAc pH 4.8 to obtain the other protein concentrations set forth in Table 1 below.

(49) The crystallization droplets were formed automatically (by an Oryx4 robot) by mixing 1 l of the protein solution with 1 l of the precipitation buffer. Each condition was repeated 8 times. Table 1 summarizes the crystallization conditions tested.

(50) TABLE-US-00002 TABLE 1 Condition Condition Condition Condition Condition A B C D E Condition F HEWL HEWL HEWL HEWL HEWL HEWL 75 mg/ml 50 mg/ml 25 mg/ml 12.25 mg/ 6.125 mg/ 3.062 mg/ml ml ml

(51) Three different plates were used, Plate 1 was left untreated in room temperature (constant 18 C.), Plate 2 was treated for 4 minutes according to the method of the present invention and then left at room temperature while Plate 3 was treated for 8 minutes according to the method of the invention and then left at room temperature. In both cases, the crystallisation solution was insulated relative to the discharge electrode.

(52) Tables 2-4 summarize the results for each from plates 1-3, respectively.

(53) TABLE-US-00003 TABLE 2 Results from plate 1 Condition A Condition B Condition C Condition D Condition E Condition F 1.sup.st crystal No crystals 1.sup.st crystal No crystals No crystals No crystals appeared 1.5 after 6 hours appeared 4 hours later days later 101 total There were 9 total crystals crystals 20 crystals appeared 1 hours later appeared 4 day later (when days later droplets were observed) 101 crystals 22 total 9 total were present crystals crystals were a week later appeared 1 present a day later week later 27 crystals were present a week later

(54) TABLE-US-00004 TABLE 3 results plate 2 Condition A Condition B Condition C Condition D Condition E Condition F 1.sup.st crystal 1.sup.st crystal 1.sup.st crystal 1.sup.st crystal No crystals No crystals appeared 1.5 appeared 3 appeared 28 appeared hours later hours later hours later 4.5 days later 176 total 27 total 14 total 3 total crystals crystals crystals crystals appeared 1 appeared 1 appeared 4 appeared 5 day later day later days later days later 176 crystals 29 crystals 14 total 3 total were a week were a week crystals were crystals were later later a week later a week later

(55) TABLE-US-00005 TABLE 4 results plate 3 Condition A Condition B Condition C Condition D Condition E Condition F 1.sup.st crystal 1.sup.st crystal 1.sup.st crystal 1.sup.st crystal No crystals No crystals appeared 0.5 appeared 2 appeared 20 appeared hours later hours later hours later 4.5 days later 225 total 55 total 18 total 11 total crystals crystals crystals crystals appeared 1 appeared 1 appeared 4 appeared 5 day later day later days later days later 225 crystals 55 crystals 18 total 11 total were a week were a week crystals were crystals were later later a week later a week later

(56) In X-ray diffraction experiments, crystals were used for data collection. A great number of datasets was obtained. Lysozyme crystals grown according to the method of the invention were qualitatively better than those obtained by the conventional vapor-diffusion method (control crystals). We used four indicators:

(57) 1) the signal intensity on the high resolution shell,

(58) 2) the mosaicity of the crystals and

(59) 3) the R.sub.merge indicator

(60) 4) the resolution

(61) The exposed crystals displayed higher values regarding of signal intensity and resolution and lower ones regarding the mosaicity and the Rmerge. Specifically, the samples exposed for 1, 2, 3 or 5 minutes displayed the highest signal intensity and resolution values combined with the lowest mosaicity and Rmerge values. The samples exposed for more than 5 minutes produced crystals of the samemore or lessquality with the control crystals.

(62) From the above experiments it is clear that, when droplets are exposed to a stream of ionized gas molecules (in this case O.sub.2.sup.1) according to the present invention in the initial stages of the crystal formation and growth process, the number of crystals increases (Tables 3 and 4), compared to control experiments (Table 2, no exposure to ionized gas-carried current) in all the different protein concentrations and the crystal diffraction quality is improved.

(63) Further, the number and the diffraction quality of the crystals formed is also increased when increasing of the time that the drops are exposed to the method of the present invention. One condition (12.25 mg/ml HEWL) provided no crystals from control droplets (without exposure to the present invention, cf. Table 1), while exposure of the droplets to the method of the invention resulted in the appearance of 3 (at 4 minutes of exposure time) and 11 (at 8 minutes of exposure time) crystals.