JP2006329775A - Observation method of compound and coloring solution used therein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an observation method of a compound, capable of certainly discriminating the crystal particles of the compound to easily perform their structural analysis, and a coloring solution used therein. <P>SOLUTION: In an X-ray diffraction experiment, the crystal image of the compound which is a sample is photographed and the X-ray irradiating position determining work of the sample is performed, while the crystal of the compound is observed on the basis of the photographed image. After the crystal of the compound has been placed in the coloring solution 6, it is scooped up along with the coloring solution 6 to be solidified and the crystal is irradiated with transmission light to photograph a crystal image. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a crystal observation method in X-ray crystal structure analysis for X-ray analysis of a crystal structure of a high molecular compound such as a protein or a low molecular compound.

  Each protein has a unique three-dimensional structure, and X-ray crystal structure analysis is one method for analyzing protein structure. Conventional X-ray crystal structure analysis was first obtained by crystallizing protein in solution. The protein crystal grains are scooped together with the solution in a cryoloop with a loop-shaped tip, and the crystal grains are put into a solution mixed with an anti-freezing agent. The measurement system is used (for example, refer to Patent Document 1).

Japanese Patent Laying-Open No. 2003-83212 (FIG. 1)

  Crystal structure analysis by X-ray is performed by irradiating crystal grains with transmitted light and photographing the crystal grains with a microscope camera while identifying the crystal grains with the naked eye, but the protein crystal grains are very small and at most 200 μm. Therefore, it was not easy to identify crystal grains with the naked eye.

  In addition, an automatic analysis system that can automatically identify crystal grains from observation images taken with a microscope camera and analyze the crystal structure has been studied, but the identification of crystal grains is still insufficient. There's a problem.

  The present invention has been made in view of such problems of the prior art, and a compound observation method capable of reliably identifying crystal grains of a compound and easily performing structural analysis thereof, and the method It aims at providing the coloring solution used for.

  In order to achieve the above object, the invention described in claim 1 of the present invention is characterized in that in X-ray diffraction experiments, a crystal image of a compound as a sample is photographed, and the sample is observed while observing the crystal from the photographed image. When performing the X-ray irradiation positioning operation, the crystal is placed in a colored solution, and then the crystal is scooped up and solidified together with the colored solution, and a crystal image is taken by irradiating the crystal with transmitted light. This is a method for observing a compound.

  The invention according to claim 2 is characterized in that the color of the colored solution and the color of the transmitted light have a complementary color relationship.

  The invention described in claim 3 is characterized in that the color of the colored solution is black and the color of the transmitted light is white.

  The invention according to claim 4 is characterized in that the colored solution is a colored anti-freezing buffer solution.

  The invention according to claim 5 is characterized in that the compound is a protein.

  The invention according to claim 6 is a colored solution used in the method for observing a compound according to any one of claims 1 to 5 and containing a crystal of the compound.

  According to the present invention, since the crystal of the compound is put in a colored solution and then the crystal is irradiated with transmitted light, the irradiated light is transmitted through the crystal portion, and the solution around the crystal is irradiated with light. It will be blocked. As a result, the edge of the crystal is emphasized and the crystal can be reliably identified, so that the crystal can be easily observed.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
FIG. 1 shows a method for observing protein crystals in an X-ray diffraction experiment of a protein. The crystal crystals are picked up by scooping up protein microcrystals grown in a buffer solution (crystallization solution) called a crystallization drop. The process until the sample is observed with a microscope camera is schematically shown.

  As shown in FIG. 1, the protein crystal to be structurally analyzed is brittle and easily broken. Therefore, the protein crystal is first grown in a crystallization drop 2 and has a thickness of 10 to 20 μm and a size of about 1 mm at the maximum. The sample is scooped with a cryoloop 4 formed of a polymer fiber yarn (for example, made of nylon), further dropped into a colored anti-freezing buffer solution 6 and then scooped with a cryoloop 4 again.

  Next, the cryoloop 4 is attached as a sample (sample) to be analyzed to a goniometer 8 that can move the cryoloop 4 in a three-dimensional direction. However, the solution scooped by the cryoloop 4 is evaporated as it is. Then, nitrogen gas for cooling is blown from the nozzle 10 onto the cryoloop 4 to solidify it into an amorphous state so that the solution does not freeze.

  Further, an illuminating device 12 and a microscope camera (CCD) camera 14 for sample observation are arranged on both sides of the goniometer 8, and the sample is moved appropriately in the three-dimensional direction by operating the goniometer 8, and from one of the samples. The illumination device 12 irradiates the transmitted light and photographs the protein crystal grains contained in the sample with the camera 14 in focus. Furthermore, the image photographed by the camera 14 is displayed on the image processing device 16, and the X-ray crystal structure analysis of the protein is performed.

  In the protein crystal structure observation method described above, the present invention is particularly characterized in that the anti-freezing buffer solution 6 is colored and the sample is irradiated with one of a plurality of colors of transmitted light. The color of the transmitted light and the color of the transmitted light to be irradiated are preferably in a complementary color relationship, which will be described in detail below.

  Protein microcrystal grains scooped by cryoloop 4 are not easily identified by the naked eye, and automatic analysis is performed to automatically identify crystal grains from observation images taken by camera 14 and perform crystal structure analysis. In the case of the system, since the position where the crystal grains are arranged becomes very important, it is indispensable to identify the protein crystal.

  In the present invention, the contrast between the crystal grains and the solution is obtained by dropping the sample in a low contrast state into the colored anti-freezing buffer solution 6 and replacing the crystallization solution surrounding the sample with the colored anti-freezing buffer solution 6. To improve the identification accuracy of crystal grains. In the antifreeze buffer solution 6, glycerol or the like was used as an antifreeze agent (antifreeze solution).

  Crystals were observed using multiple colors of transmitted light and multiple colors of anti-freezing buffer solution, and it was investigated whether the combination of colors had effects such as edge enhancement of crystals in the cryoloop.

  First, the anti-freezing buffer solution was colored with any of red, blue, green, and black ink. The protein crystals are scooped from the crystallization solution with a cryoloop, immersed in a colored antifreeze buffer solution for several seconds, again scooped up with the antifreeze buffer solution again with a cryoloop, and attached to the experimental apparatus. Cooled down. In addition, any of red, blue, green, and white was set as transmitted light, and an image was taken with a CCD camera to observe the appearance of the crystal.

The proteins, buffer solutions, etc. used in this example were as follows.
-Protein sample: Crystal of egg white lysozyme-Buffer solution (crystallization solution): aqueous solution of sodium chloride 1M + sodium acetate 50 mM-Antifreeze buffer solution: Liquid in which glycerol at a volume ratio of 27 percent is mixed as an antifreeze agent -Colored dye: Each color water-based ink with a volume percentage of several percent of the anti-freezing buffer solution (collected from a commercially available sign pen)

  2 and 3 show sample images when a red anti-freezing buffer solution is used. FIG. 2 shows the first time, and FIG. 3 shows the second time. 2 (a) and 3 (a) show green transmitted light, FIGS. 2 (b) and 3 (b) show red transmitted light, and FIGS. 2 (c) and 3 (c) show green transmitted light. FIGS. 2D and 3D are sample images when white transmitted light is used and blue transmitted light is used.

  4 and 5 show sample images when a blue anti-freezing buffer solution is used. FIG. 4 shows the first time (with epi-illumination), and FIG. 5 shows the second time. 4 (a) and 5 (a) show the green transmitted light, FIGS. 4 (b) and 5 (b) show the red transmitted light, and FIGS. 4 (c) and 5 (c) show the red transmitted light. FIGS. 4D and 5D are sample images when white transmitted light is used and blue transmitted light is used.

  Further, FIG. 6 shows a sample image when there is epi-illumination when a green anti-freezing buffer solution is used, FIG. 6 (a) shows green transmitted light, and FIG. 6 (b) shows red transmitted light. FIG. 6C is a sample image when white transmitted light is used.

  7 to 9 show sample images when a black anti-freezing buffer solution is used. FIG. 7 shows the first time, FIG. 8 shows the second time, and FIG. 9 shows the third time. . 7 (a), 8 (a) and 9 (a) show red transmitted light, and FIGS. 7 (b), 8 (b) and 9 (b) show white transmitted light. FIG. 9C shows a sample image when green transmitted light is used, and FIG. 9D is a sample image when blue transmitted light is used.

As a result of checking these sample images, the following conclusions were obtained.
(1) When black anti-freezing buffer solution and white transmitted light are used, the buffer region in the cryoloop does not transmit light, and only the crystal transmits light. Easy to see.
(2) The appearance of crystals also changes depending on the combination of the intensity of transmitted light and the shade of the antifreeze buffer solution. If the transmitted light is strong and the color of the antifreeze buffer solution is dark, the crystals are emphasized and easily visible.
(3) Regardless of the color of the anti-freezing buffer solution and transmitted light, if there is epi-illumination, the buffer surface will shine and crystals will be difficult to see.
(4) In a state where the anti-freezing buffer solution is spherical and the crystal is thickly covered with the anti-freezing buffer solution, or in a state where the cryoloop is sideways, it is difficult to identify the crystal. Conversely, if the cryoloop faces the front and there is little anti-freezing buffer solution, it is easy to observe.

In addition, the following can also be used as a buffer solution, an antifreezing agent, and a coloring dye.
Buffer solution: Crystallizing solution in general (usually water + pH buffering agent + precipitating agent, adjustment conditions differ for each sample)
・ Anti-freezing agent: Sugar reagents such as sucrose, glucose, xylitol, organic solvents such as polyethylene glycol, ethylene glycol, methylpentadiol, ethanol, methanol ・ Coloring dyes: Water-soluble inks and pigments in general (as anti-freezing buffer solution) , When using an aqueous solution in which an antifreezing agent is dissolved in a buffer solution), or
Pigments such as oil-based inks (when mineral oil is used as an anti-freezing buffer solution)

  In the above embodiment, the case where the crystal structure of the protein is analyzed has been described as an example. However, the present invention is not limited to the analysis of the crystal structure of the protein. It can also be applied to crystal structure analysis of compounds and the like.

  In the method for observing the compound according to the present invention, the crystal is solidified and photographed after the crystal is placed in a colored solution, so that the edge of the crystal is emphasized and can be reliably identified, such as a protein. It is useful for crystal structure analysis of high molecular weight compounds or other low molecular weight compounds.

It is the schematic which shows the protein crystal observation method in the X-ray-diffraction experiment of protein. It is the photograph of the sample image at the time of using a red anti-freezing buffer solution, (a) is green transmitted light, (b) is red transmitted light, (c) is white transmitted light, (d ) Uses blue transmitted light. It is the photograph of another sample image at the time of using a red anti-freezing buffer solution, (a) is green transmitted light, (b) is red transmitted light, (c) is white transmitted light, (D) uses blue transmitted light. It is a photograph of a sample image when there is epi-illumination when a blue anti-freezing buffer solution is used, (a) is green transmitted light, (b) is red transmitted light, (c) is white Transmitted light and (d) use blue transmitted light, respectively. It is the photograph of another sample image at the time of using a blue anti-freezing buffer solution, (a) is green transmitted light, (b) is red transmitted light, (c) is white transmitted light, (D) uses blue transmitted light. It is a photograph of a sample image when there is epi-illumination when a green anti-freezing buffer solution is used, (a) shows green transmitted light, (b) shows red transmitted light, and (c) shows white light. Each transmitted light is used. It is the photograph of the sample image at the time of using a black anti-freezing buffer solution, (a) is using the red transmitted light, (b) is using the white transmitted light, respectively. It is the photograph of another sample image at the time of using a black anti-freezing buffer solution, (a) is using the red transmitted light, (b) is using the white transmitted light. It is the photograph of another sample image at the time of using a black anti-freezing buffer solution, (a) is red transmitted light, (b) is white transmitted light, (c) is green transmitted light. , (D) use blue transmitted light.

Explanation of symbols

2 Crystallization drop,
4 Cryoloop,
6 anti-freezing coloring buffer,
8 Goniometer,
10 Cooling nitrogen gas nozzle,
12 Lighting device,
14 Sample observation microscope camera,
16 Image processing apparatus.

Claims (6)

In an X-ray diffraction experiment, a crystal image of a compound as a sample was taken, and when performing X-ray irradiation positioning work of the sample while observing the crystal with the taken image,
A method for observing a compound, comprising: putting a crystal in a colored solution, scooping the crystal together with the colored solution and solidifying the crystal, and irradiating the crystal with transmitted light to take a crystal image. The method for observing a compound according to claim 1, wherein the color of the colored solution and the color of the transmitted light have a complementary color relationship. The method for observing a compound according to claim 1, wherein the color solution is black and the color of the transmitted light is white. The method for observing a compound according to any one of claims 1 to 3, wherein the colored solution is a colored anti-freezing buffer solution. The method for observing a compound according to any one of claims 1 to 4, wherein the compound is a protein. A colored solution used in the method for observing a compound according to any one of claims 1 to 5 and containing a crystal of the compound.

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