ARRAY, SOAKING SOLUTIONS AND METHOD OF SELECTING SOAKING CONDITIONS FOR SMALL MOLECULES IN BIOLOGICAL MACROMOLECULAR CRYSTALS

Abstract

Subject-matter of the present invention is an array comprising soaking solutions for soaking a biological macromolecular crystal. Further, the subject of the invention is a rule-based method of selecting specific soaking solution compositions having a specific composition comprising composite solute(s), water (w), crystallization solution (crs) and/or organic solvent(s). Additionally, the subjects matter of the invention the soaking solutions obtained by the method of the invention and a screening method for small molecules comprising molecular probes, fragments and drug-size molecules using the soaking solutions and the use of the soaking solutions in a screening method for small molecules on a macromolecular crystal.

Claims

1. A method of selecting the composition of a soaking solution for soaking the crystal form of a biological macromolecule wherein the soaking solution comprises an organic solvent (os), a compatible solute (cs), and a crystallization solution (crs) and/or water (w); said method comprising; 1) preparing a first series of in to xn individual soaking solutions by mixing os, cs, crs and w wherein: a) x is at least 2 b) each soaking solutions of the first series comprises the same organic solvent (os), the same compatible solute (cs), and the same crystallization solution (crs), and c) V.sub.os and V.sub.cs are the same in each solution, or, alternatively, the number of moles compatible solute Ncs per volume organic solvent V.sub.os is the same in each solution, and d) Vcrs and Vw are changed in each solution, and in the solution in, V.sub.crs is the minimum V.sub.crs (V.sub.crs min) and the V.sub.w is the maximum V.sub.w (V.sub.wmax), and in the solution xn, V.sub.w is the minimum V.sub.w (V.sub.wmin) and the V.sub.crs is the maximum V.sub.crs(V.sub.crs max), and when x>2, in any additional solutions between in and xn, the V.sub.crs is varied between V.sub.crs min and V.sub.crs max and, inversely, Vw is varied between V.sub.wmin and V.sub.wmax 2) transferring at least one crystal of the biological macromolecular crystal per compartment into x compartments, each compartment comprising one of the solutions prepared in step 1); or transferring each solution prepared in step 1) into x compartments, each compartment comprising at least one crystal of the biological macromolecular crystal from its crystallization solution. 3) controlling the crystal in each compartment 4) selecting the soaking solution(s) which is/are suitable for soaking the crystal; and wherein the compatible solute is selected from polyols, amino acids, methylamines, and mixtures thereof, and wherein the organic solvent is selected from liquid carbohydrates, protic or aprotic, of low reactivity, that can serve to solve small molecules and, mixtures thereof, and wherein the crystallization solution crs is obtained from the crystallization process of said crystal or is equivalent thereto in terms of osmotic net pressure, permittivity, solubility of the according biological macromolecule, and effect on its structural conformation, comprising pH and buffer materials, additives, and precipitants.

2. The method according to claim 1, wherein said soaking solution comprises a crystallization solution (crs) or an equivalent aqueous salt solution thereto and an organic solvent (os) and a compatible solute (cs), wherein said compatible solute (cs) is a polyol a or a mixture thereof, and said organic solvent (os) is a liquid carbohydrate protic or aprotic, of low reactivity, or a mixture thereof, that can serve to solve small molecules, more preferably, said compatible solute (cs) is a poly(oxyethylene) and said organic solvent (os) is selected from a (alkylated) sulfoxide, cyclic non-aromatic ether, straight or branched chain monohydric aliphatic alcohol, alkylated formamide or a mixture thereof, most preferably, the compatible solute (cs) is selected from poly(oxyethylene)s polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600 or a mixture thereof and said organic solvent (os) is selected from dimethyl sulfoxide, 1,4-dioxane, dimethylformamide, methanol, ethanol, or a mixture thereof and wherein the ratio of volume compatible solute Vcs to volume organic solvent Vos is the same and the volume ratio is 1:1000000 to 1000000:1, preferably between 8:1 to 1:8 and more preferably between 3:1 to 1:1 (Vcs:Vos); and wherein the ratio of volume crystallization solution or equivalent aqueous salt solution Vcrs to the combined volumes of organic solvent Vos and compatible solute Vcs is 1:1000000 to 1000000:1, preferably between 99:1 to 1:99 and most preferably between 85:15 to 1:1 (Vcrs: (Vos; Vcs)).

3. The method according to claim 1, wherein said soaking solution comprises a crystallization solution (crs) or an equivalent aqueous salt solution thereto and an organic solvent (os) and a compatible solute (cs), wherein said compatible solute (cs) is a polyol or a mixture thereof, and said organic solvent is a (alkylated) sulfoxide, cyclic non-aromatic ether, straight or branched chain monohydric aliphatic alcohol, alkylated formamide, or a mixture thereof, more preferably, said compatible solute (cs) is a polyhydric alcohol or a mixture thereof and said organic solvent (os) is selected from a (alkylated) sulfoxide, cyclic non-aromatic ether, straight or branched chain monohydric aliphatic alcohol, alkylated formamide or a mixture thereof, most preferably, the compatible solute (cs) is selected from the polyhydric alcohols propane-1,2,3-triol (glycerol), ethane-1,2-diol (ethylene glycol), 2-Methylpentane-2,4-diol (2-Methylpentane-2,4-diol),(3R,4S,5S,6R)-2-(2,3-dihydroxypropoxy)-6-(hydroxymethyl)oxane-3,4,5-triol (1-Glucosylglycerol) or a mixture thereof, and said organic solvent (os) is selected from dimethyl sulfoxide, 1,4-dioxane, dimethylformamide, methanol, ethanol, or a mixture thereof and wherein the ratio of volume compatible solute Vcs to volume organic solvent Vos is the same and the volume ratio is 1:1000000 to 1000000:1, preferably between 8:1 to 1:8 and more preferably between 3:1 to 1:1 (Vcs:Vos); and wherein the ratio of volume crystallization solution or equivalent aqueous salt solution Vcrs to the combined volumes of organic solvent Vos and compatible solute Vcs is 1:1000000 to 1000000:1, preferably between 99:1 to 1:99 and most preferably between 85:15 to 1:1 (Vcrs: (Vos; Vcs)).

4. The method according to claim 1, wherein said soaking solution comprises a crystallization solution (crs) or an equivalent aqueous salt solution thereto and an organic solvent (os) and a compatible solute (cs), wherein said compatible solute (cs) is a polyol a or a mixture thereof, and said organic solvent (os) is a liquid carbohydrate protic or aprotic, of low reactivity, or a mixture thereof, that can serve to solve small molecules, more preferably said compatible solute (cs) is a mono-, di-, tri, oligo- or polysaccharide or a mixture thereof and said organic solvent (os) is selected from a (alkylated) sulfoxide, cyclic non-aromatic ether, straight or branched chain monohydric aliphatic alcohol, alkylated formamide or a mixture thereof, most preferably, the compatible solute cs is selected from the saccharides (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxane-3,4,5-triol (trehalose) or β-D-Fructofuranosyl α-D-glucopyranoside (sucrose) or a mixtures thereof, and said organic solvent (os) is selected from dimethyl sulfoxide, 1,4-dioxane, dimethylformamide, methanol, ethanol, or a mixture thereof and wherein the molar ratio of compatible solute to organic solvent is the same and the molar ratio is 1:10000 to 10000:1, preferably between 100:1 to 1:100 and more preferably between 10:1 to 1:10, and wherein the ratio of volume crystallization solution or equivalent aqueous salt solution Vcrs to the combined volumes of organic solvent Vos and compatible solute Vcs is 1:1000000 to 1000000:1, preferably between 100:1 to 1:100 and most preferably between 85:15 to 1:1 (Vcrs: (Vos; Vcs)).

5. The method according to claim 1 wherein said soaking solution comprises a crystallization solution (crs) or an equivalent aqueous salt solution and an organic solvent (os) and a compatible solute (cs), wherein said compatible solute (cs) is a methylamine or a mixture thereof and said organic solvent (os) is liquid carbohydrate protic or aprotic, of low reactivity, or a mixture thereof that can serve to solve small molecules, more preferably said compatible solute (cs) is selected from N,N-dimethylmethanamine oxide (Trimethylamine N-oxide or TMAO), 2-trimethylammonioacetate (trimethylglycine or betaine), 2-(Methylamino)acetic acid (N-methylglycine or sarcosine), or a mixture thereof and said organic solvent (os) is a (alkylated) sulfoxide, cyclic non-aromatic ether, straight or branched chain monohydric aliphatic alcohol, alkylated formamide, or a mixture thereof, most preferably said compatible solute (cs) is selected from N,N-dimethylmethanamine oxide (Trimethylamine N-oxide or TMAO), 2-trimethylammonioacetate (trimethylglycine or betaine), 2-(Methylamino)acetic acid (N-methylglycine or sarcosine), or a mixture thereof and said organic solvent (os) is selected from dimethyl sulfoxide, 1,4-dioxane, dimethylformamide, methanol, ethanol, or a mixture thereof and wherein the molar ratio of organic solvent (os) to compatible solute (cs) Mos/Mcs is the same and the molar ratio Mos/Mcs is 1:10000 to 10000:1, preferably between 100:1 to 1:100 and more preferably between 10:1 to 1:10, and wherein the ratio of volume crystallization solution or equivalent aqueous salt solution Vcrs to the combined volumes of organic solvent Vos and compatible solute Vcs is 1:1000000 to 1000000:1, preferably between 100:1 to 1:100 and most preferably between 85:15 to 1:1 (Vcrs: (Vos; Vcs)).

6. The method according to claim 1, wherein said soaking solution comprises a crystallization solution (crs) or an equivalent aqueous salt solution thereto and an organic solvent (os) and a compatible solute (cs), wherein said compatible solute (cs) is polyethylene glycol 400 and said organic solvent (os) is dimethyl sulfoxide, wherein the ratio of volume Vcs polyethylene glycol 400 to volume Vos dimethyl sulfoxide is the same and the volume ratio is 1:1000000 to 1000000:1, preferably between 8:1 to 1:8 and more preferably between 3:1 to 1:1 (Vcs: Vos); and wherein the ratio of volume crystallization solution or equivalent aqueous salt solution Vcrs to the combined volumes of the organic solvent (os) dimethyl sulfoxide and the compatible solute (cs) polyethylene glycol 400 is 1:1000000 to 1000000:1, preferably between 99:1 to 1:99 and most preferably between 85:15 to 1:1 (Vcrs: (Vos:Vcs)).

7. The method according to claim 1, wherein said soaking solution comprises a crystallization solution (crs) or an equivalent aqueous salt solution thereto and an organic solvent (os) and a compatible solute (cs), wherein said compatible solute (cs) is ethane-1,2-diol (ethylene glycol) and said organic solvent (os) is dimethyl sulfoxide, wherein the ratio of volume Vcs ethane-1,2-diol (ethylene glycol) to volume Vos dimethyl sulfoxide is the same and the volume ratio is 1:1000000 to 1000000:1, preferably between 8:1 to 1:8 and more preferably between 3:1 to 1:1 (Vcs: Vos); and wherein the ratio of volume crystallization solution or equivalent aqueous salt solution Vcrs to the combined volumes of the organic solvent (os) dimethyl sulfoxide and the compatible solute (cs) ethane-1,2-diol (ethylene glycol) is 1:1000000 to 1000000:1, preferably between 99:1 to 1:99 and most preferably between 85:15 to 1:1 (Vcrs: (Vos:Vcs)).

8. The method according to claim 1, wherein said soaking solution comprises a crystallization solution (crs) or an equivalent aqueous salt solution thereto and an organic solvent (os) and a compatible solute (cs), wherein said compatible solute (cs) is propane-1,2,3-triol (glycerol) and said organic solvent (os) is dimethyl sulfoxide, wherein the ratio of volume Vcs propane-1,2,3-triol (glycerol) to volume Vos dimethyl sulfoxide is the same and the volume ratio is 1:1000000 to 1000000:1, preferably between 8:1 to 1:8 and more preferably between 3:1 to 1:1 (Vcs: Vos); and wherein the ratio of volume crystallization solution or equivalent aqueous salt solution Vcrs to the combined volumes of the organic solvent (os) dimethyl sulfoxide and the compatible solute (cs) propane-1,2,3-triol (glycerol) is 1:1000000 to 1000000:1, preferably between 99:1 to 1:99 and most preferably between 85:15 to 1:1 (Vcrs: (Vos:Vcs)).

9. The method according to claim 1, wherein said soaking solution comprises a crystallization solution (crs) or an equivalent aqueous salt solution thereto and an organic solvent (os) and a compatible solute (cs), wherein said compatible solute (cs) is 2-methylpentane-2,4-diol (MPD) and said organic solvent (os) is dimethyl sulfoxide, wherein the ratio of volume Vcs 2-methylpentane-2,4-diol (MPD) to volume Vos dimethyl sulfoxide is the same and the volume ratio is 1:1000000 to 1000000:1, preferably between 8:1 to 1:8 and more preferably between 3:1 to 1:1 (Vcs: Vos); and wherein the ratio of volume crystallization solution or equivalent aqueous salt solution Vcrs to the combined volumes of the organic solvent (os) dimethyl sulfoxide and the compatible solute (cs) 2-methylpentane-2,4-diol (MPD) is 1:1000000 to 1000000:1, preferably between 99:1 to 1:99 and most preferably between 85:15 to 1:1 (Vcrs: (Vos:Vcs)).

10. The method according to claim 1m further comprising 1) preparing m series of in to yn individual soaking solutions by mixing os, cs, crs and w wherein m is at least 1 and wherein in each of the m series a) y is at least 2 b) os, cs and crs are the same as in the first series, and c) V.sub.os is the same as in the first series, and d) V.sub.cs is varied and different from the V.sub.cs of the first series, or, alternatively, Ncs is varied and different from Ncs of the first series; and wherein in each individual solution of each of the m series e) Vos and Vcs are conserved, or, alternatively, Vos and Ncs are conserved, and f) Vcrs and Vw are changed in each solution, and in the solution in, V.sub.crs is the minimum V.sub.crs (V.sub.crs min) and the V.sub.w is the maximum V.sub.w (V.sub.wmax), and in the solution yn, V.sub.w is the minimum V.sub.w (V.sub.wmin) and the V.sub.crs is the maximum V.sub.crs(V.sub.crs max), and when y>2, in any additional solutions between in and yn, the V.sub.crs is varied between V.sub.crs min and V.sub.crs max and, inversely, V.sub.w is varied between V.sub.wmin and V.sub.wmax 2) transferring at least one crystal of the biological macromolecular crystal per compartment into y compartments, each compartment comprising one of the solutions prepared in step 1); or transferring each solution prepared in step 1) into y compartments, each compartment comprising at least one crystal of the biological macromolecular crystal from its crystallization solution.

11. The method according to claim 1, further comprising the 1) preparing p series of in to zn individual soaking solutions by mixing os, cs, crs and w wherein p is at least 1 and wherein in each of the p series a) z is at least 2 b) os, cs and crs are the same as in the first series, and c) V.sub.cs is the same as in the first series, or, alternatively, Ncs is the same as in the first series, and d) V.sub.os is varied and different from the V.sub.os of the first series; and wherein in each individual solution of each of the p series e) V.sub.os and V.sub.cs are conserved, or, alternatively, V.sub.os and Ncs are conserved, and f) Vcrs and V.sub.w are changed in each solution, and in the solution in, V.sub.crs is the minimum V.sub.crs (V.sub.crs min) and the V.sub.w is the maximum V.sub.w (V.sub.wmax), and in the solution zn, V.sub.w is the minimum V.sub.w (V.sub.wmin) and the V.sub.crs is the maximum V.sub.crs(V.sub.crs max), and when z>2, in any additional solutions between in and zn, the V.sub.crs is varied between Vcrsmin and Vcrsmax and, inversely, Vw is varied between V.sub.wmin and V.sub.wmax 2) transferring at least one crystal of the biological macromolecular crystal per compartment into z compartments, each compartment comprising one of the solutions prepared in step 1); or transferring each solution prepared in step 1) into z compartments, each compartment comprising at least one crystal of the biological macromolecular crystal from its crystallization solution.

12. The method according to claim 1, wherein an equivalent aqueous salt solution different from the crystallization solution is used, and wherein the salt selected from disodium;2,3-dihydroxybutanedioate (sodium tartrate), (2R,3R)-2,3-dihydroxybutanedioate (dipotassium tartrate), azanium; phosphates (ammonium phosphates), diazanium; sulfate (ammonium sulfate), triazanium;2-hydroxypropane-1,2,3-tricarboxylate (ammonium citrate), sodium; acetate (sodium ethanoate), azanium; acetate (ammonium acetate), dilithium; sulfate, lithium; chloride, lithium; acetate, lithium; formate, lithium; nitrate, sodium; nitrate, sodium; chloride, potassium; chloride, sodium; formate, monophosphate from sodium and potassium, di- and polyphosphates from sodium and potassium, sodium citrates (sodium citrates as sodium dihydrogen 2-hydroxypropane-1,2,3-tricarboxylate (monosodium citrate), disodium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate (disodium citrate), trisodium 2-hydroxypropane-1,2,3-tricarboxylate (trisodium citrate)), magnesium; diformate, magnesium; dichloride, magnesium; sulfate, calcium; dichloride; dihydrate, disodium; butanedioate (sodium succinate), cadmium(II) sulfate (cadmium sulfate), disodium; propanedioate (sodium malonate), magnesium; diacetate (magnesium acetate), zinc; diacetate (zinc acetate), calcium; diacetate (calcium acetate) and wherein the salt for the equivalent solution is preferably selected from lithium; acetate, lithium; chloride, lithium; formate, lithium; nitrate, dilithium; sulfate, sodium; chloride, potassium; chloride, magnesium; dichloride, diazanium; sulfate (ammonium sulfate), sodium; acetate (sodium ethanoate), sodium citrates (sodium citrates as sodium dihydrogen 2-hydroxypropane-1,2,3-tricarboxylate (monosodium citrate), disodium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate (disodium citrate), trisodium 2-hydroxypropane-1,2,3-tricarboxylate (trisodium citrate), magnesium; diformate, sodium; nitrate.

13. The method according to claim 1, wherein crs, os, cs and/or w contain small molecules or preferably os contains small molecules to be analyzed, which diffuse into the biological macromolecular crystal.

14. A method of small molecule screening for a biological macromolecular crystal, comprising performing the screening in a soaking solution selected according to the method of claim 13.

15. A soaking solution obtainable by the method of selecting the composition of a soaking solution according to claim 1.

16. A method of small molecule screening for a biological macromolecular crystal, comprising performing the screening in a soaking solution according to claim 15.

17. An array arranged to perform the method according to claim 1, wherein said array comprises a first dimension of at least two individual soaking solutions (1n to xn) and a second dimension of at least two individual soaking solutions (1m to ym) wherein each of said soaking solutions is located in a separated compartment of said array, and wherein each of said soaking solution comprises an organic solvent (os), a compatible solute (cs), a crystallization solution (crs) and water, and wherein the ratio of volume compatible solute Vcs to volume organic solvent Vos is the same within a series of soaking solutions in said first dimension, or, alternatively, the number of moles compatible solute Ncs per volume organic solvent Vos is the same within a series of soaking solutions in said first dimension, and wherein the individual soaking solutions of said first dimension comprise the same organic solvent and the same compatible solute within a series of said soaking solutions in the first dimension, respectively, and wherein the ratio of the volume of water Vw and the volume of the crystallization solution Vcrs is varied over the individual soaking solutions of the series in the first dimension and wherein one of the individual soaking solutions of the series in the first dimension has a minimal or zero Vcrs and a maximum Vw and another individual soaking solution of said first dimension has a minimal or zero Vw and a maximum Vcrs and wherein the other individual soaking solutions of the series in said first dimension take values of Vcrs and Vw in between the two before-mentioned values.

18. A method of obtaining a soaking solution comprising using an array according to claim 17.

Description

FIGURE DESCRIPTION

[0520] FIG. 1: 1A is a picture of a screening plate used for soaking selection; FIG. 1B shows the details of one of the well of a 24 well plate and on FIG. 1C a schematic representation of the well during an soaking experiment is depicted.

[0521] FIG. 2 shows a preparation scheme according to the method of the invention for selection of soaking solutions comprising CS: compatible solute; OS—organic solvent; R (or crs)-reservoir solution (or crystallisation solution); W—water. The lines (solute A-D) represent the first dimension and the columns (1-6) the second dimension. The top line is the first series. The volume of CS (V.sub.cs) is changed in each series and the V.sub.w and V.sub.R (or Vcrs) are changed in each solution within each individual series. The volume of os (Vos) is the same for each series

[0522] FIG. 3 shows a preparation scheme according to the method of the invention for selection of soaking solutions Like in FIG. 2, the volume of CS (V.sub.cs) is changed in each series and the V.sub.w and V.sub.R (V.sub.crs) are changed in each solution within an individual series. The volume of os (Vos) is the same for each series but is different from the V.sub.os of the series/plate of FIG. 2. The plate of FIG. 2 can overlap the plate of FIG. 3: this would constitute the third dimension according to the invention.

[0523] FIG. 4 shows an example of a scheme according to the method of the invention for selection of soaking solutions. The V.sub.cs is changed in each series but the V.sub.os remains 10% in all solutions

[0524] FIG. 5 shows an example of a scheme according to the method of the invention for selection of soaking solutions. The V.sub.cs is changed in each series but the V.sub.os remains 20% in all solutions. The plate of FIG. 4 can overlap the plate of FIG. 5: this would constitute the third dimension according to the invention.

[0525] FIG. 6 shows an example of a scheme according to the method of the invention for selection of soaking solutions. The V.sub.cs is changed in each series but the V.sub.os remains 30% in all solutions. The plate of FIG. 6 can overlap the plate of FIGS. 5 and 6: this would constitute the third dimension according to the invention.

[0526] FIG. 7 shows another example of a scheme according to the method of the invention for selection of soaking solutions. The CS is composed of a mix of compatible solutes and EB refers to a freshly prepared salt solution of NaCl that exerts an equivalent role as crs as the original crs does.

[0527] FIG. 8 is an example of a scheme according to the method of the invention for selection of soaking solutions and example 2. The crs is not from a solution of the crystallisation process of the crystal. In this example, the CRS is an equivalent buffer” (EB). The cs is a mixture of compatible solutes e.g. PEG3350 (50% w/v) and PEG400 in a ratio of 35:25 the EB is 1 M NaCl solution and the OS is 10% DMSO. The volume of CS (V.sub.cs) is changed in each series and the Vw and Vcrs (VNaCl) are changed in each solution within an individual series. The volume of os (V.sub.os=10%) is the same for each series of the plate.

[0528] FIG. 9 is an example of a scheme according to the method of the invention for selection of soaking solutions and example 2. The crs is not from a solution of the crystallisation process of the crystal. In this example, the CRS is an equivalent buffer“(EB). The cs is a mixture of compatible solutes e.g. PEG3350 (50% w/v) and PEG400 in a ratio of 35:25 the EB is 1 M NaCl solution and the OS is 20% DMSO. The volume of CS (V.sub.cs) is changed in each series and the Vw and Vcrs (VNaCl) are changed in each solution within an individual series. The volume of os (V.sub.os=20%) is the same for each series of the plate but different from the series/plate of FIG. 8. The plate of FIG. 9 can overlap the plate of FIG. 8: this would constitute a third dimension according to the invention.

[0529] FIG. 10A shows an example of the visual difference between suitable crystal (left) and crystals suffering from a severe quality loss (right) due to detrimental soaking conditions/solution. FIG. 10B shows a template for the X-Ray inspection of crystals.

[0530] FIG. 11: shows the criteria used for visual inspection and to control and select the solutions having minimal impact on the crystal

[0531] FIG. 12 shows an image of a diffraction pattern after an X-Ray experiment.

[0532] FIG. 13 shows a template for the X-Ray inspection of crystals.

[0533] FIG. 14 shows the criteria used for X-Ray inspection and to control and select the solutions having minimal impact on the crystal.

[0534] FIG. 15: shows the results of the visual inspection followed by X-Ray diffraction of crystals from different proteins TGT, Lysozyme, Endothiapepsin, IspD, Thermolysine and aldose reductase II after 24 h in the tested soaking solutions of FIG. 8. The best solutions have been selected and are highlighted (3rd line, column 3 and 5, and 4th line column 4). Dark grey-crystallization conditions that demonstrated suitable for all tested types of protein crystals. Medium grey-Conditions that destroyed at least one crystal type from the tested protein crystal types.

[0535] FIG. 16: shows the results of the visual inspection followed by X-Ray diffraction of crystals from different proteins (TGT, Lysozyme, Endothiapepsin, IspD, Thermolysine, aldose reductase II) after 24 h in the tested soaking solutions of FIG. 9. The best solutions have been selected and are highlighted (1st line, column 4 and 3rd line column 3). Dark grey-crystallization conditions that demonstrated suitable for all tested types of protein crystals. Medium grey-Conditions that destroyed at least one crystal type from the tested protein crystal types.

[0536] FIG. 17 shows the pipetting scheme for one of the 24-well experimental plates used for the small molecule fragment screening of hCAll crystal of example 2.

[0537] FIG. 18 shows an image of a diffraction pattern after a X-Ray experiment on hCAll crystal.

[0538] FIG. 19 shows an example of a potential workflow representing the different step of the processes of the invention performed in a soaking selection experiment with subsequent fragment screening.

[0539] FIG. 20 shows a 3D-model of hCAll representing the protein and an overlay of all identified binders distributed over different locations on the protein. The magnification in the circle shows the crowd of ATP-pocket binders in more detail.

[0540] FIG. 21 shows the excerpt of a reconstructed electron density.

[0541] FIG. 22 shows the same as FIG. 21 but completed with the protein's amino acid sequence of hCAll and structurally fixed water molecules (small sphere) as well as Znll cofactor (big sphere).

[0542] FIG. 23: shows the same as FIG. 22 but with highlighted (ellipse) unassigned electron density that is representative for a small molecule fragment-hit.

[0543] FIG. 24 shows the same as FIG. 23 but completed with the representation of the respective small molecule fragment in the formerly unassigned electron density highlighted in FIG. 23.

[0544] FIG. 25 shows the excerpt of the structure elucidating the binding mode in terms of hydrogen bonds (dotted lines; numbers represent the length of the hydrogen bonds) of the selected small molecule fragment to the amino acid side chains of the hCAll protein and to structurally fixed water (dot). The big sphere is a Zn″-cofactor.

EXAMPLES

Example 1: Preparation of an Experiment for the Determination of Soaking Solution Compositions for Protein Crystal

[0545] Crystals, that have been prepared beforehand, were harvested via a cryo-loop and transferred to the wells of an experimental plate (arrays) (see example on FIG. 1) that was prepared and distributed in two dimensions according to the method of the invention (FIGS. 2 and 3).

[0546] Each line is a series, the top line is the first series. Each series comprises 6 solutions (1-6).

[0547] On FIG. 2, in the first series, Vcrs max is 80% and Vcrs min is 0% while VWmax is 80% and VW min is 0%. VT is 100%. In each of the following series (lines 2-4), Vwmax and Vcrsmax are 70, 60 and 50% respectively, The V.sub.os (10%) is the same in all solutions of all series but the V.sub.cs changes in all series compared to the first series but remains the same for all solutions within each series.

[0548] On FIG. 3, in the first series, Vcrs max is 70% and Vcrs min is 0% while Vwmax is 70% and V.sub.wmin is 0%. VT is 100%. In each of the following series (lines 2-4), Vwmax and Vcrs max are 60, 50 and 40% respectively,

[0549] Compared to FIG. 2, the V.sub.os of FIG. 3 is 20% and is the same in all solutions of all series of FIG. 3. However, in FIG. 3, the V.sub.cs changes in all series compared to the first series but remains the same for all solutions within each series of FIG. 3.

[0550] Each line of each plates is a 1.sup.st dimension, each column creates a second dimension. If the plates of FIG. 2 and FIG. 3 are overlapped, this creates a 3.sup.rd dimension according to the invention.

[0551] FIGS. 4-6 shows another example of arrays according to the invention. The V.sub.os is the same for all solutions within a single plate. The V.sub.os is 10%, 20%, 30% for the plates of FIGS. 4, 5 and 6 respectively. The Vcrs (designated R in the FIGS. 4-6 is the same in each series of each single plates but varies between the plates from 30% for the plate of FIG. 4, 25% for FIGS. 5 and 20% for FIG. 6.

[0552] Example 2—Screening of 96 Fragments Using Human Carbonic Anhydrase II-Protein (hCAll)

[0553] 2.1 Crystallization of hCAll

[0554] Crystallization of human carbonic anhydrase II (hCAll) were conducted at 18° C. from a mixture of a solution A containing 2.7 M ammonium sulfate and 0.1 M TRIS at pH 7.8 which was saturated with para-chloromercuribenzoic acid and a solution B containing protein at a concentration of 10 mg/ml in the original expression buffer containing 0.05 M TRIS, pH 7.8. The wells of 24-well experimental plates (Hampton Research) for hanging drop crystallization were equipped with 0.5 ml of solution A as reservoir buffer. Then, 2 μl of the crystallization solution containing solutions A and B were mixed on siliconized cover slips (Jena Bioscience) and placed on each well, with silicon grease as sealant. Under this vapor diffusion crystallization approach crystals grew within one day.

[0555] Crystals of the following proteins were also prepared following a similar method for subsequent use in controlling the solutions prepared according to the method described below: tRNA-guanine transglycosylase (TGT), lysozyme, endothiapepsin2-C-methyl-Derythritol4-phosphate cytidylyltransferase (IspD), thermolysine, aldose reductase II).

[0556] 2.2 Determination of Soaking Parameters

[0557] Subsequently, the hCAll-crystals were subjected to a subset of soaking solution compositions that was derived from an extensive screening according to the present invention on a number of different crystals that were previously grown in very different crystallization solutions. The screening was performed using NaCl as “equivalent” crs instead of the original crystallization solution used to grow the crystal in the above crystallization of hCAII.Furthermore, a mixture of PEG400 and PEG3350 (50%) was used instead of a single cs. FIGS. 8 and 9 show an excerpt from the pipetting scheme according to the present invention. The solutions were prepared according to the method of the invention as follows:

a) Array/Plate of FIG. 8

[0558] Preparation of a first series of 6 individual soaking solutions by mixing os, w and crs(NaCl) and mixture of cs.

[0559] The series with the lowest volume of CS was used as the benchmark or first series and was prepared with 40% of cs. Each soaking solutions of the first series comprises the same organic solvent (os) (DMSO) and the same mix of compatible solute (cs) and the V.sub.os 10% (1 0 μl) and V.sub.cs 40% (4 μl) remains the same in all solutions of the series. However, the volume/proportion of NaCl (crs) and Vw are changed in each solution of this first series, and one of the solution is composed of Vcrsmin is 5% (0,5 μl) and Vwmax is 45% (4,5 μl) (see column 1), and another solution is composed of Vwmin is 20% (2 μl) and the Vcrsmax is 30% (3 μl) (see column 6) and in the other solutions (see columns 2-5) the Vw is varied between Vwmin and Vwmax and, inversely, Vcrs is varied between Vcrsmin and Vcrsmax. In this example, Vcrs is incrementally varied between Vcrsmin and Vcrsmax and, inversely, Vw is incrementally varied between Vwmin and Vwmax in any additional solutions (see column 2-5) and in any additional solutions, the increment of Vw between Vwmin and Vwmax and of Vcrs between Vcrsmin and Vcrsmax remains the same, 5% (0,5 I) between each solution of the first series. [0560] The solutions were distributed in the wells of a line (line on FIG. 8) of an array thus constituting a first dimension. The solutions were preferably distributed following an order from the lowest Vcrs to the highest Vcrs but the order of the distribution in the array can be random.

[0561] Then or simultaneously, the further steps were performed as follows [0562] Preparation of an additional series of 6 individual soaking solutions by mixing os, w, crs (NaCl) and cs. In this example of FIG. 8, the number of individual soaking solutions 6 is conserved and is the same as the number of individual soaking solutions of the benchmark series. In the additional series (line 2-4), os and V.sub.os are the same as in the first series but the V.sub.cs is changed (line 2: 50%, line 3: 55% and line 4: 60%)). Within the additional series, each soaking solutions of the first/benchmark series comprises the same organic solvent (os) (DMSO) and the same mixture of compatible solute (cs) and the V.sub.os remains the same in all solutions of the series. However, the volume/proportion of Vcrs (CB) and Vw are changed in each solution of each additional series. Within each additional series, the volume/proportion of NaCl (crs) and Vw are changed in each solution, and one of the solution is composed of Vcrsmin is 5% (0,5 μl) and Vwmax is 45% (4,5 μl) (see column 1), and another solution is composed of Vwmin is 20% (2 μl) and the Vcrsmax is 30% (3 μl) (see column 6) and in the other solutions (see columns 2-5) the Vw is varied between Vwmin and Vwmax and, inversely, Vcrs is varied between Vcrsmin and Vcrsmax. In this example, Vcrs is incrementally varied between Vcrsmin and Vcrsmax and, inversely, Vw is incrementally varied between Vwmin and Vwmax (see column 2-5), the increment of Vw between Vwmin and Vwmax and of Vcrs between Vcrsmin and Vcrsmax remains the same, 5% (0,5 μl) between each solution.

[0563] In this example, the number of individual soaking solutions i.e. 6 the same as the number of individual soaking solutions of the first series and the increment of Vcrs between Vcrsmin and Vcrsmax and of Vw between Vwmin and Vwmax is the same in the first series and in the additional series.

[0564] The solutions were distributed in the wells of additional line (line 2-4 on FIG. 8) of an array thus constituting a second dimension. The solutions were preferably distributed following an order from the lowest Vcrs to the highest Vcrs but the order of the distribution in the array can be random.

b) Array/Plate of FIG. 9

[0565] Simultaneously or not, the following steps were performed as follows: [0566] Preparation of an additional series of 6 individual soaking solutions by mixing os, w and crs(NaCl) and mixture of cs.

[0567] The series with the lowest volume of CS of FIG. 8 is still used as the benchmark or first series and was prepared with 40% of cs.

[0568] The further steps were performed as follows [0569] Preparation of an additional series of 6 individual soaking solutions by mixing os, w, crs (NaCl) and cs. In this example of FIG. 9, the number of individual soaking solutions 6 is conserved and is the same as the number of individual soaking solutions of the benchmark series. In the additional series (line 1 cs-mix=40%), the os, the cs and the V.sub.cs are the same as in the first series. However, the V.sub.os is changed (line 2: 20% (2 μl) compared to the benchmark series.

[0570] Within the additional series, the volume/proportion of NaCl (crs) and Vw are changed in each solution, and one of the solution is composed of Vcrsmin is 5% (0,5 μl) and Vwmax is 35% (3,5 μl) (see column 1), and another solution is composed of Vwmin is 10% (1 μl) and the Vcrsmax is 30% (3 μl) (see column 6) and in the other solutions (see columns 2-5) the Vw is varied between Vwmin and Vwmax and, inversely, Vcrs is varied between Vcrsmin and Vcrsmax. In this example, Vcrs is incrementally varied between Vcrsmin and Vcrsmax and, inversely, Vw is incrementally varied between Vwmin and Vwmax (see column 2-5), the increment of Vw between Vwmin and Vwmax and of Vcrs between Vcrsmin and Vcrsmax remains the same, 5% (0,5 I) between each solution.

[0571] The solutions were distributed in the wells of additional line (line 1 on FIG. 9) of a separate array thus constituting a third dimension. Indeed if the plate of FIG. 8 and FIG. 9 were overlapped, the lines and column of each plate would constitute a second dimension and the top line of the plate of FIG. 8 and the top line of the plate of FIG. 9 would overlap and constitute a third dimension. The solutions were preferably distributed following an order from the lowest Vcrs to the highest Vcrs but the order of the distribution in the array can be random.

[0572] Following the same rules, the solutions of the additional series (line 2: 50%, line 3: 55% and line 4: 60% of cs) can be prepared. In this example, the series of FIG. 8 (second line) with 50% of CS mixture would be the benchmark series for the additional series of FIG. 9 with the same V.sub.cs (line 2 of FIG. 9) or vice versa. The same rule applies for the series of the lines 3 (V.sub.cs: 55%) and 4 (V.sub.cs:60%) of the plates of the FIGS. 8 and 9. Importantly, the method can be implemented even when the number of solutions in the additional series compare to the benchmark series and/or the Vcrs and Vw minimum and maximum are different. Similarly, the increment of Vcrs between Vcrsmin and Vcrsmax and of Vw between Vwmin and Vwmax can be the same (as in this example) or different in the first series and in the additional series.

[0573] Following the same principles, another plate with solutions of V.sub.os=25% was also prepared (figure not shown.

c) Control and Selection of the Suitable Soaking Solution(s)

[0574] For selection of the solutions which are/is suitable for soaking a biological macromolecule crystal, crystals from different proteins (for example tRNA-guanine transglycosylase (TGT), lysozyme, endothiapepsin, 2-C-methyl-Derythritol4-phosphate cytidylyltransferase (IspD), thermolysine, aldose reductase II) were placed in each compartments of the array of FIGS. 8 and 9. The crystals were placed under microscope for control by visual analysis followed by X-ray diffraction when possible e.g. when the crystal was still present and not completely destroyed by the solution.

[0575] This study revealed some solution compositions that were suitable for soaking several and even all individual crystal types.

[0576] FIG. 15: highlights the soaking solutions that, according to visual inspection followed by X-Ray diffraction, were suitable for all crystals after 24 h in the tested soaking solutions of FIG. 8. The best solutions have been selected and are highlighted (3rd line, column 3 and 5, and 4th line column 4). Dark grey—crystallization conditions that demonstrated suitable for all crystals from different proteinsMedium grey-Conditions that destroyed at least one crystal type from the range of tested protein crystal types.

[0577] FIG. 16: highlights the soaking solutions that, according to visual inspection followed by X-Ray diffraction, were suitable for all crystals after 24 h in the tested soaking solutions of FIG. 9. The best solutions have been selected and are highlighted (1st line, column 4 and 3rd line column 3). Dark grey—crystallization conditions that demonstrated suitable for all crystals from different proteins. Medium grey-Conditions that destroyed at least one crystal type from the range of tested protein crystal types.

[0578] The solutions selected as being suitable for soaking the crystal of all protein types included in this study are reported in the table I below. The solution 6 was selected from a plate not shown where the V.sub.os in all solutions of all series is 25%.

[0579] The soaking solutions selected according to the method of the invention are considered to be of universal character in the sense that they can be suitably used for soaking different types of crystals.

[0580] Crystals of hCAll were subjected only to these resulting 6 soaking solutions. The control experiments (visual and X-ray) showed that these soaking solutions were suitable also for hCAll. The best result was obtained for solution 4 in Table 1. The crystals from this soaking solution composition showed the very best diffraction quality in X-Ray-tests at a synchrotron with a resolution of 0.97 Å and a mosaicity of 0.105°.

[0581] Therefore, solution 4 was selected as soaking solution for a subsequent small molecule fragment screening on hCAll crystals.

TABLE-US-00001 TABLE I 1 2 3 4 5 6 NaCl(1M) : 1.5 μl NaCl(1M): 1.5 μl NaCl(1M): 2.5 μl NaCl(1M): 2.0 μl NaCl(1M): 2.0 μl NaCl(1M): 2.5 μl 3350(50%): 3.0 μl 3350(50%): 3.0 μl 3350(50%): 3.0 μl 3350(50%): 3.5 μl 3350(50%): 2.0 μl 3350(50%): 2.5 μl PEG400: 2.5 μl PEG400: 2.5 μl PEG400: 2.5 μl PEG400: 2.5 μl PEG400: 2.0 μl PEG400: 2.5 μl DMSO: 1.0 μl DMSO: 2.0 μl DMSO: 1.0 μl DMSO: 1.0 μl DMSO: 2.0 μl DMSO: 2.5 μl H.sub.2O: 2.0 μl H.sub.2O: 1.0 μl H.sub.2O: 1.0 μl H.sub.2O: 1.0 μl H.sub.2O: 2.0 μl

[0582] 2.3 Fragment Screening on hCAll

[0583] For the small molecule fragment screen, four experimental plates with 24 wells each were equipped according to the selected soaking solution 4 of table I composition comprising of 35% (v/v) aqueous PEG3350 solution (50% w/v), 20% (v/v) sodium chloride solution 1.0 M, 25% (v/v) PEG400, 10% (v/v) water and 10% (v/v) DMSO. Instead of DMSO alone, 1 M fragment was dissolved in DMSO (end concentration in each well 100 mM) (FIG. 17). Afterwards, freshly prepared crystals were transferred to the wells using cryo-loops and were incubated at 18° C. Overall, the experiment subjected hCAll crystals to 96 different small molecule fragments. After 15 h (in some cases after 3 minutes) of soaking time the soaked crystals were harvested using cryo-loops and vitrified in liquid nitrogen. Data collection took place at a synchrotron. Data indexing, integration, and scaling were performed using the software tools XDS and XDSAPP2.0. Model building was carried out using Coot and refinements were performed in Phenix.

[0584] 2.4 Results

[0585] Data evaluation yielded 8 binders out of 96 of resolutions of 0.95-1.68 Å. The refinement of according data yielded 3D-models of the interaction of these 8 fragments with hCAll at different interaction sites. Four binders were active site binders, three of which directly coordinate to hCAll's Znll cofactor, one indirectly via a water molecule. Two of these binders were hydrazides, a chemotype that has not been reported to inhibit carbonic anhydrases so far. The hydrazides contain phenyl rings which are involved in a π-interaction with the side chain of a leucine (Leul98). The respective coordination geometry allows for a hydrogen bond between the hydrazide group's Nβ and the threonine's side chain hydroxy function (Thr199). As a result, the phenyl moiety is orientated differently in comparison to hCAll's prototypical inhibitor benzenesulfonamide (BSA) while triggering a flip of the sidechain of before mentioned leucine (Leu198), which is also observable upon the interaction of hCAll with N-hydroxybenzamide. Another binder, a sulfonamide, bound with its expected conventional geometry. The fourth active-site binder, salicylic acid, binds as other hydroxybenzoicacids do: by an interaction of its carboxylate function with the ZnII-bound hydroxide ion/water molecule via a hydrogen bond. Furthermore, it interacts with a threonine's side chain (Thr200), in a fashion similar to a previously described interaction between BSAs and hCAll. Furthermore, the screening revealed four binders at three further interaction sites (FIG. 20). One of these interaction sites turned out to establish covalent interactions with two of the respective remote binders. These additional binding sites can be addressed by small molecules which possibly can exert some relevant effect on the protein's biological activities. Their pharmacological meaning emerges if one succeeds to link these findings to biological data. The structural data can now be analysed in terms of binding modes of the small molecule fragments to the protein. FIG. 25 shows an example of a small molecule fragment binder in the active site in direct vicinity to the Znll-cofactor (big sphere). The image shows 3 hydrogen bonds of which one is established with a water molecule.

[0586] The study is one example of a very successful implementation of an embodiment of the present invention to determine soaking parameters for a small molecule fragment screening. That proves that the technology according to the present invention is a very successful means to perform rapid and reliable small molecule-screenings and delivers high-quality data. This data is subsequently either exploited in order to identify promising follow-up compounds that are more drug-size, and which already exist, or it is exploited by medicinal chemists to introduce modifications of the small molecule fragment implemented by chemical synthesis. Such follow-up compounds are again validated crystallographically and assays for evaluation of according biological activity.

Example 3

[0587] In order to find soaking solution compositions for macromolecular crystals for example for a protein of interest the experimenter must first obtain suitable crystals (step a in workflow in FIG. 19). The crystallization is conducted, for example, according to the vapor diffusion approach in commercially available 24-well sitting drop crystallization plates (or any other plate format)(FIG. 1A). For this purpose, the reservoirs of each 24-well plate used are filled with a buffer according to a beforehand established crystallization protocol. Furthermore, a crystallization drop is prepared in the indentation on top of the column of each of the wells by adding some volume of a protein solution to some volume of the buffer, that is also used in the reservoir. Usually, but not always the volumes are in a ratio of 1:1 (FIGS. 1B and 1C). The resulting protein concentration is usually, but not always, in the range of 2 to 30 mg/ml but can also reach concentrations of 250 mg/ml or more. Afterwards the wells are sealed using an adhesive film, so called sealing tape. All these steps are conducted in an according to the crystallization protocol correctly tempered laboratory, usually at 4° C., 10° C., 18° C., 20° C. The plates are stored on a shelf in this laboratory for the time needed for crystallization, usually a few days to a few weeks.

[0588] After the crystallization process is finished, the method of selection of the invention is conducted in order to map conditions for a small molecule fragment screening (same temperature). In an example, the indentations of three 24-well crystallization plates (or any other plate format) are provided with possible soaking solutions according to the present invention as is exemplified in FIG. 2-9 (step b of workflow in FIG. 19). These experiments are usually but not always conducted at the same temperature as for the crystallization. The experimental setup aims at identifying soaking solution compositions that stabilize crystals of the POI in a quality maintaining manner. For this purpose, a crystallization buffer or a salt solution is varied in a first series and distributed in a first dimension by mixing with water (see lines 5% compatible solute A on FIG. 4-5-6 for instance).

[0589] On this “first”, also referred to as benchmark series, the V.sub.cs and V.sub.os remains constant. The Vcrs min is 30% in column 1 and the Vcrs max is 80% in column 6 while the Vw max in column 1 is 55% and the Vw min in column 6 is 5%.

[0590] The V.sub.cs or a mixture from more than one compatible solute is varied in a second dimension. The V.sub.cs of each of the series is different from the V.sub.cs of the other series (lines 2-4) and of the first series (1st line) (see lines 10%, 15% and 20% of compatible solute A in FIG. 4-6). However, in each series the V.sub.os remains unchanged and the Vcrs and Vw vary inversely between Vcrs min, Vw max (1st column) and Vcrs max, Vw min (column 6).

[0591] Finally, the same rule based method is used to prepare the plate of FIG. 4 and the plates of FIGS. 5 and 6. The main variant/difference between the plates of FIGS. 4-6 is that the V.sub.os remains the ase in each series of the same plate but is varied on each plate i.e. in a third dimension (FIG. 4: 10%, FIG. 5: 20%, or FIG. 6: 30%).Thus, a broad spectrum of organic solvent—compatible solutes-ratios are balanced against different buffer compositions in order to determine compositions that compensate for the individual component's detrimental effects.

[0592] Subsequently, the film used for sealing the 24-well plates is removed from the plates used for crystallization of the POI. The meanwhile grown crystals are removed from their growth solutions using a special laboratory tool usually equipped with a nylon loop for crystal picking or any other appropriate tool for the so-called “crystal fishing” and transferred to the possible soaking solutions in the indentations of the two new 24-well plate that were prepared according to an instantiation of the method of the invention (step c of workflow in FIG. 19). These plates are also sealed using sealing tape.

[0593] The purpose of the next steps is to evaluate the crystals quality after exposure to the solutions prepared above, the maximal soaking time and a proper relation between soaking time and crystal quality. Within a certain time, usually the next 24 hours, pictures of each indentation are taken under a microscope at least 1 time, usually three times, sometimes more, under a microscope in order to document the crystal's condition for example immediately after the transfer, after 1 hour and after 24 hours (FIG. 10A). In parallel, usually notes are taken in order to supplement the information in the pictures. (A template for such notes is exemplified in FIG. 10B). Notes refer to different types of cracks crystals can show (longitudinal, lateral, crisscross), to the appearance of the crystals surface (e.g. smooth, rough) or edges (e.g. sharp, roundish) and their behavior in polarized light (strong color, some color, no color) (FIG. 11). In the most cases not all of the crystals survive in all the conditions. Some crystals dissolve over time or are damaged while other crystals are in good condition. Therefore, a x-ray evaluation of the crystal's quality is obligatory (step d of workflow in FIG. 19). For this purpose, the sealing tape is removed from the plate in order to transfer the remaining crystals from the plate into liquid nitrogen or another vitrification medium. In case that the x-ray study is conducted at room temperature, previous vitrification is not required. The crystals' vitrification takes place in the nylon loops mentioned before or any other equivalent transfer-equipment like litho-loops or capillaries. The vitrified crystals in the nylon loops or other transfer-equipment are stored in so called “cryo vials” under liquid nitrogen or another vitrification medium until they are subjected to a x-ray examination. Crystals can also be stored in any other appropriate storage containers as, for example, Uni-Pucks.

[0594] For the x-ray examination the crystals in the nylon loops or other transfer-equipment are removed from the cryo-vials or other storage containers and mounted on a x-ray machine in a so called cryo-stream that maintains the cryogenic temperature around the protein crystal. In case of x-ray studies at room temperature, no cryo-stream is required. Afterwards, x-ray data is collected. Using an inhouse x-ray source two to several pictures are collected. The raw data (FIG. 12) is evaluated regarding resolution, and mosaicity, the appearance of artifacts like data originating from unwanted ice crystals in the protein crystal and sometimes a phenomenon called “twinning” (FIGS. 13 and 14). In case of data collected at a synchrotron, the appearance of the so called “electron density” is judged by an experienced crystallographer as suitable or not suitable. Based on this data a decision is made on which soaking condition or conditions is/are to be chosen for an small molecule fragment screening (step e/g of workflow in FIG. 19). In contrast to traditional optimizations of soaking solutions which result in one single soaking condition, the exemplified method usually results in a spectrum of soaking conditions. From this spectrum the experimenter can choose alternative soaking solutions to face detrimental effects on the crystal that may be caused by for example special properties of small molecule fragments or pH changes etc.

[0595] Optionally, at this stage of the workflow stress tests can be conducted (step f of workflow in FIG. 19). For this purpose, one or more of the previously obtained soaking solutions is/are selected and prepared in a new 24-well plate. Afterwards, crystals are transferred to these solutions. Instead of pure DMSO, molecular probes or small molecule fragments for stress tests often dissolved in DMSO are used. They exert different kinds of stress such as pH-stress or osmotic stress on the crystals. Based on the subsequently performed data evaluation (same as in step d of workflow in FIG. 19) one or more final soaking solutions can be chosen.

[0596] Usually, a decision is made for one soaking condition which is subsequently used for the subsequent small molecule fragment screening. For a small molecule fragment screening comprising 300 small molecule fragments 13 of the 24-well plates are prepared by pipetting the respective soaking solution composition of the chosen soaking solution without DMSO. Instead of pure DMSO, 300 small molecule species at concentrations of usually 1 M dissolved in DMSO are used and added to the prepared 300 wells. This results in final concentrations of the small molecule fragments in each soaking solution of usually 100 mM but may be higher or lower (step h of workflow in FIG. 19). Following that, usually two newly prepared crystals (sometimes just one or more than two) are transferred to each soaking solution and soaked for usually 24 hours (step i of workflow in FIG. 19). Then, the crystals are fished, transferred to liquid nitrogen for vitrification (step j of workflow in FIG. 19), stored in cryo-vials, Uni-pucks or some other applicable storage device and sent to a synchrotron facility, where data collection takes place (step k of workflow in FIG. 19). In case of x-ray studies at room temperature no vitrification is required.

[0597] In data collection individual data sets for all crystals are obtained comprising of several hundreds to thousands of pictures taken at different angles showing reflections of the x-ray beams (exemplified in FIG. 18). These reflections stem from interactions between x-rays and electrons and, therefore, can be exploited to reconstruct the so-called electron densities. Such electron densities define the space in the crystals where electrons are located and are representative for the protein's geometry. Using computational methods important information are extracted from these data sets. To reconstruct the geometry further information is needed, the so-called phases, that are not extractable from this kind of experiment without heavy atom derivatization. This information can be derived in an approach called molecular replacement from previously obtained structural solutions of the same or very similar proteins. The result is a 3-dimensional model of the protein's geometry according to the positions of all the electrons in the protein. This electron density is merged with the known sequence of amino acids of the respective protein and positions of structurally fixed water molecules are identified. This solution for the structure is a model that must be optimized in several structure refinement cycles in order to obtain a convincing fit between the measured data and the model. The result is an atomically resolved representation of the geometry of the target (step I of workflow in FIG. 19).

[0598] Each of these structures is examined in order to identify electron density (FIG. 21-24) that can be assigned to the special small molecule fragment that was used for soaking of this very crystal (FIG. 24). If this is the case, this small molecule fragment is judged as a so-called “hit”. Examination of all data sets reveals all small molecule fragment-binders. Data sets without additional electron density that is assignable to respective small molecule fragments are discarded. Further refinement of each of these “hit”-data sets reveal the exact geometry of the interaction between the protein and the respective small molecule fragments (FIG. 25). Therefore, the described process provides validated knowledge about small molecule fragments that bind to the protein target and delivers structurally elucidated binding modes that can be used for medicinal-chemical optimization processes. Instead of small molecule fragments also more drug-size molecules can be used for example to validate follow-up compounds resulting from medicinal-chemical optimization. Furthermore, special molecular probes can be used to map properties of the binding sites or to obtain information about putative pharmacophore patterns etc.