JPH08338818A - Sample mounting fixture for X-ray analysis - Google Patents

Abstract

(57) [Abstract] [Purpose] A sample such as a protein crystal can be axially mounted without applying stress, and no strong diffraction lines are generated from the mounting tool itself, and a blind area is generated. To provide a sample mount for X-ray analysis. [Structure] Rod 31 made of an amorphous material such as fused quartz
Cylindrical member 30 made of an amorphous material such as fused quartz
Is mounted so that the axis of the cylindrical member 30 is substantially perpendicular to the axis of the rod 31. [Effect] Since the sample mount portion is cylindrical and the sample is exposed, the cryogen can be brought into direct contact with the sample and rapidly frozen. Since the sample adheres to the relatively wide flat wall surface of the cylindrical member, the crystal can be axially mounted without being distorted. Also, because it is made of an amorphous material,
No strong diffraction line is generated from the mounting tool itself.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sample mount for X-ray analysis, and more particularly to a sample for X-ray analysis suitable for crystallographic analysis of a biopolymer crystal at a low temperature such as liquid nitrogen. Regarding mounting equipment.

[0002]

[Prior Art] Protein X-ray crystal structure analysis has made various contributions to the elucidation of the three-dimensional structure, principle of construction, and reaction mechanism of protein molecules that are responsible for chemical reactions that support life in vivo, and its importance will be increasing in the future. It is thought that it will increase more and more. However, a problem that is always troublesome for those performing X-ray diffraction experiments on protein crystals is deterioration of crystallinity due to radiation damage of protein crystals. Although the details of the radiation damage mechanism of protein crystals have not been fully elucidated, the degree of radiation damage is strongly correlated with the total X-ray dose irradiated to protein crystals and the dose per unit time. Are known. X-rays absorbed by protein crystals are converted into energy and heat of radical generation, which eliminates the interaction between protein molecules in the crystals, which is thought to result in crystallinity degradation and dissolution. . For example, 12ke
X-rays having V energy of 0.1 at a rate of 10 ¹⁰ / sec.
When incident on a crystal having a linear absorption coefficient of 19 mm ^-1 ,
Assuming that all absorbed X-rays are converted to heat, the temperature rise of the crystal is estimated to be about 1 mK / sec.

[0003] As a method of suppressing radiation damage, a short-wavelength X-ray (0.03
2) or a method in which a diffraction experiment is performed at a low temperature to quickly remove generated heat and suppress radical generation. The former is premised on the use of a large synchrotron radiation experimental facility, so diffraction experiments at low temperatures have been desired and tried in practice, considering laboratory-level measurements.

[0004] The biggest difficulty in conducting a diffraction experiment at low temperatures is that water contained in a large amount in protein crystals precipitates as hexagonal ice. Ice precipitation destroys protein crystals from within. To prevent ice precipitation during the cooling process,
Replacing the crystal mother liquor of the protein crystal with one containing a cryoprotectant,
Further, upon cooling, it is essential to rapidly and efficiently cool the protein crystals by directly exposing them to a cryogen. In view of such demands, the capillary tubes conventionally used in protein X-ray crystal structure analysis experiments cannot expose proteins mounted therein directly to a cryogen, so that they can be used at low temperatures. It is not suitable as a crystal mounting tool for performing X-ray diffraction experiments.

As a mounting tool suitable for rapidly cooling protein crystals, a mounting tool using a thin quartz plate as shown in FIG. 2A is known [Acta Cryst. B45 , 190].
-199 (1989)]. This mounting tool has a thickness of about 100 μm and a surface area of about 500 × 500 μm ² at the tip of the quartz rod 21.
Is assembled by fixing the thin quartz plate 22 of FIG. 1 with an adhesive 23, and the protein crystals in the crystal mother liquor are
2 and attached to the surface. FIG. 2 (b)
As shown in the figure, platinum, tungsten having a diameter of about 80 μm,
Microfine metal wire 25 made of constantan or the like by bending the loop and secured with an adhesive 26 to the distal end of the quartz rod 24 mounted device are also known [J. Appl. Cryst. 2
3 , 387-391 (1990)]. This mounting tool mounts the crystal on the ultrafine metal wire 25 using the surface tension of the crystal mother liquor. Each of the mounting tools shown in FIG. 2 is used with the lower ends of the rods 21 and 24 fixed to a goniometer in an X-ray diffractometer.

[0006]

However, in the mounting device using the thin quartz plate, slight irregularities on the surface of the thin quartz plate 22 cause small mechanical deformation of the protein crystal, and the obtained X-ray diffraction data is obtained. It may impair reliability. In addition, an operation of scooping the crystal in the crystal mother liquor and placing it on a thin quartz plate and an operation of subsequently removing the crystal mother liquor are required, and there is a drawback that the handling is lacking in speed.

[0007] In a mounting tool using an ultrafine metal wire, as shown in the schematic cross-sectional view of FIG. 3, the crystal 27 is indicated by an arrow due to the surface tension of a crystal mother liquor 28 existing in a gap between the wire 25 and the protein crystal 27. Strong force acts on the crystal lattice, and correct diffraction data may not be obtained. Further, the more serious problem is that a so-called blind region is generated due to the use of a metal wire, and when an X-ray hits a loop made of a metal wire, a strong diffraction scattering pattern generated from the loop enters the detector, and This makes it impossible to measure the intensity of the inverse space existing in such an arrangement. In addition, since the loop wire has a circular cross section, there is a problem that it is difficult to orient the crystal even when the protein crystal is sufficiently strong.

It is an object of the present invention to provide a new crystal mount for low-temperature X-ray diffraction which has solved the above-mentioned problems of the prior art.

[0009]

SUMMARY OF THE INVENTION A sample mounting tool used for low-temperature X-ray structure analysis of protein crystals and the like is described in (1).
High sample cooling efficiency, (2) no mechanical damage to the mounted sample crystal, (3) simple mounting of the sample, (4) strong diffraction lines from the mounting tool itself It is desirable to satisfy the following conditions: (5) the crystal can be oriented, (6) the thermal expansion coefficient is small, the crystal is not broken by rapid cooling, and (7) the X-ray can be transmitted. .

In order to satisfy the above conditions, X according to the present invention is used.
The sample holder for line analysis is characterized in that a cylindrical member made of an amorphous material is fixed to the tip of a rod made of an amorphous material such that the axis of the cylindrical member is substantially perpendicular to the axis of the rod. And The rod and the cylindrical member can be made of fused quartz or soda glass. The cylindrical member can be manufactured by cutting a capillary tube made of, for example, fused quartz into a ring. Further, the cylindrical member may be a cylindrical body whose cross section perpendicular to its axis is circular,
A cylindrical body of another shape whose cross section perpendicular to the axis is a triangle or a square may be used.

[0011]

The sample mounting portion, such as a protein crystal, is cylindrical and the sample is exposed, so that the cryogen can be brought into direct contact with the sample and rapidly frozen. Since the sample adheres to the relatively wide flat wall surface of the cylindrical member, it can be mounted without distorting the crystal. The sample can be mounted on the wall of the cylindrical member by a simple operation of immersing the tip of the mounting tool in the sample liquid and scooping out the crystal sample in the crystal mother liquor, and then removing the liquid. No special operation is required. That is, in order to scoop up the crystal sample in the crystal mother liquor into the internal space of the cylindrical member, the crystal surface of the crystal is likely to coincide with the surface created by the surface tension of the crystal mother liquor with respect to the cylindrical member,
By repeatedly performing the scooping operation, mounting with the crystal axis aligned in a predetermined direction can be easily performed. For example, in the case of bipyramidal crystals which are often found in protein crystals, as shown in FIG. 8 (a), it is easy to mount with two vertices positioned inside the cylindrical member, and in the case of a rectangular parallelepiped crystal, As shown in FIG. 8B, it is easy to mount with one of the crystal faces in close contact with the wall surface of the cylindrical member.

Since the cylindrical member for mounting the sample is made of an amorphous material such as fused quartz or soda glass, no strong diffraction lines are generated from the mounting tool itself. In addition, fused quartz and soda glass do not break even when they are rapidly cooled by contact with a cryogen, and have a sufficient X-ray transmittance.
Table 1 below shows a comparison between the performance of the sample mounting tool according to the present invention and the performance of a sample mounting tool using a conventional thin quartz plate or a fine metal wire. Table 1 Sample mounting tool The present invention Thin quartz plate Ultra-fine metal wire ワ イ ヤ Material Fused quartz Fused quartz Fine metal wire Cooling efficiency Good Good Good Mechanical load on the crystal No Yes Yes Blind No No Yes Shafting Yes Yes No As described above, the sample mounting fixture for X-ray analysis according to the present invention can be used for samples such as protein crystals. Satisfies all the conditions necessary for low-temperature X-ray structural analysis of the sample, and by using this sample mount, highly reliable X-ray diffraction data can be easily obtained.

According to the protein X-ray crystal structure analysis using the sample mounting device for X-ray analysis according to the present invention, it is possible to carry out the structural analysis of the intermediate state of the protein at low temperature,
It can also contribute to the elucidation of how the thermal oscillations of the protein itself and the dynamics of the water molecules surrounding it have a correlation with the function of the protein.

[0014]

The present invention will be described below in detail with reference to examples. First, a method for manufacturing a sample mounting device will be described. As shown in the cross section in FIG. 1A, a groove 12 is formed on the surface of a synthetic resin plate 11, and an outer diameter of 200 to 1000 is formed in the groove.
A fused silica capillary tube 13 having a thickness of 10 μm and a thickness of 10 μm was inserted and fixed. Then, as shown in FIG. 1 (b), the same cutting teeth 15 as those used for cutting chips from a wafer in the field of manufacturing semiconductor parts are fed at a feed speed of 0.5 mm / sec, and the synthetic resin plate is fed. A cut was made in the surface of 11 and the capillary tube 13 was cut at a plurality of locations. Thus, as shown in FIG.
A fused quartz cylindrical member 30 having a height of 100 to 200 μm and a thickness of about 10 μm was produced.

[0015] As shown in FIG.
Was bonded to the tip of a glass fiber 31 using an epoxy resin 32. This epoxy resin retains its adhesive performance even in quick freezing using liquid ethane,
Even when the cylindrical member 30 itself was immersed in liquid ethane at a speed of 1 m / sec, it was not destroyed by the impact and temperature change. The length of the glass fiber 31 is appropriately determined according to the size of the sample mount of the X-ray diffraction apparatus so that the irradiation X-ray passes through the position of the cylindrical member 30. The mounting tool 40 thus manufactured is further used by bonding it to a metal pin 34 such as brass. The metal pin 34 is adhered to a samarium-cobalt magnet 35 having a diameter of 5 mm and a thickness of 2 mm so that positioning is easy in rapid cooling with low-temperature nitrogen gas.

FIG. 6 schematically shows an X-ray diffraction apparatus using the sample mounting device of the present invention. The mounting tool 40 having the crystal sample mounted on the cylindrical member at the tip is attached to the goniometer 50 by the magnet 35. The sample is constantly cooled during the measurement by the low-temperature nitrogen gas flowing down from the low-temperature gas blowing device 51. X-rays generated from an X-ray generator 52 such as a rotating anti-cathode type X-ray generator are monochromated using a nickel filter, converged by an X-ray focusing mirror 53, and irradiated to a sample held on a mount 40. Is done. The diffraction X-ray pattern generated from the sample is detected by a photoconductor 54 such as an imaging plate.

Hereinafter, a measuring method using the X-ray diffractometer of FIG. 6 will be described. A difficulty in collecting protein crystal data at low temperatures is that the water occupying most of the solvent region of the protein crystal is frozen as hexagonal ice with cooling. In order to inhibit the formation of hexagonal ice during the initial cooling and freeze water into a glassy state, it is necessary to infiltrate the cryoprotectant into the crystal mother liquor and increase the cooling rate of the crystals. Water molecules in the protein are frozen into a glass by the combination of cryoprotectant and quick freezing. The cryoprotectant has an effect on the phase transition of water, such as lowering the equilibrium freezing point, suppressing the uniform growth of ice, and increasing the supercooling phenomenon. As a cryoprotectant to be penetrated into protein crystals, methanol, glycerol, 2-methyl-2,4-pentanediol, saccharose, polyethylene glycol and the like can be used. In that case, it is effective to set the concentration to 15% (w / v) or more.

Next, a measurement example will be described. Bart
Unik et al. [J. Mol. Biol. 210 , pp. 813-829 (198
9)], low-density-filled bovine trypsin orthorhombic crystals were prepared. The crystal mother liquor was 2.1 M ammonium sulfate, 1% (w / v) benzamidine, 1 mM calcium chloride, 20 mM sodium cacodylate (pH
6.0). In addition, 25% (w /
v) Saccharose was added, and the solvent was replaced by dialysis with 12
Time went. Thereafter, the trypsin crystal sample was mounted on the mounting tool 40 shown in FIG.

Trypsin crystal sample is 250 × 150 × 4
The cylindrical member 3 of the mounting tool has a size of 00 μm ³ and the crystal axis is oriented vertically as schematically shown in FIG.
It was frozen at 0 and mounted. The mounting tool on which the crystal sample was mounted was mounted on a goniometer 50 of an X-ray diffractometer as shown in FIG. 6, and the temperature was maintained at 100 K by blowing a low-temperature nitrogen gas from a low-temperature gas blowing device 51. CuKα radiation (wavelength 0.141818) from the X-ray generator 52 operated at 50 kV and 80 mA
The light is condensed by the line condensing mirror 53, and the cylindrical member 30 of the mounting tool is condensed.
The trypsin crystal sample mounted on the sample was irradiated and measured by the vibration crystal method. Table 2 shows the measurement results. Table 2 Lattice constant a = 6.33 b = 6.26 c = 6.80 nm Resolution 4.6 to 0.18 nm R _merge ^I 0.049 Vibration angle 2 degrees / plate Exposure time 45 minutes / plate Total measurement angle 90 degrees In Table 2, R _merge ^I is defined by the following equation (1), and is a quantity serving as an index indicating how accurate the measurement was.

[0020] ^{_{R merge I = Σ h Σ i}} | I i (h) - <I (h)> | / Σ h Σ i I i in (h) (1) where, h is the exponent of the reciprocal lattice points (3 Dimensional vector), and I (h) is the reflection intensity (integrated intensity) of the index h. In the measurement, the intensity of a desired set of h (usually about 10,000) is measured, but in the vibration crystal method, a plurality of desired sets of I (h) can be measured. Now, assuming that n I (h) can be measured, they are represented as follows, and the average is represented as <I (h)>
And

I ₁ (h), I ₂ (h),..., I _n (h) Accordingly, the numerator of R _merge ^I is the sum of deviations from the average value of each reflection intensity at all measurement points. , The denominator is the sum of the reflection intensities for all measurement points. As can be seen from the above definition, the smaller the value of R _merge ^I , the higher the statistical accuracy, which is zero in an ideal experiment, but is 0.06 in a normal experiment at room temperature in a laboratory.
From 0.07% to 0.07%. The size of the above crystal is relatively small in consideration of data collection in a laboratory, but the exposure time can be sufficiently obtained by the low-temperature experiment using the mounting tool of the present embodiment, and radiation damage is reduced. R _merge ^I shows 0.049, which is a good value.

According to another experiment of the present inventors, a crystal which can only collect data at a resolution of 0.6 nm due to radiation damage in a measurement at room temperature was subjected to a low-temperature experiment using the crystal mounting device of the present invention. The resolution could be greatly extended to 0.25 nm. Rapid freezing may be performed by flash freezing with a slush of liquid ethane, liquid propane, or the like. Liquid nitrogen is not preferable because it has a low cooling rate due to its foaming property. Further, once the cooling is completed, the temperature is measured while constantly cooling using a low-temperature nitrogen gas spraying device, so that the temperature is lowered from the liquid nitrogen temperature by 2%.
Diffraction intensity data can be collected at any temperature up to about 20K.

The above-mentioned mounting device, which is usually made by cutting a capillary tube used in an experiment at room temperature into a thin ring, can transmit X-rays in all directions, and uses the wall surface. By doing so, there is an advantage that the crystal axis of the crystal can be aligned with respect to the incident X-ray beam. Techniques for collecting diffraction intensity data for protein X-ray crystal structure analysis at low temperatures have been used in both laboratories and experiments using synchrotron radiation X-rays.
It is important in reducing radiation damage of protein crystals due to X-rays. This technique allows biopolymer crystals to be exposed to X-rays for extended periods of time, resulting in statistically reliable diffraction intensity data. Furthermore, by applying this technique, it becomes possible to investigate physical properties of proteins, such as the temperature dependence of internal vibrations of proteins and the role of water molecules bound to proteins.

[0024]

According to the X-ray sample mounting device of the present invention,
A sample such as a protein crystal can be axially mounted without applying stress to the sample. In addition, clear diffraction data can be obtained because no strong diffraction line is generated from the mounting tool itself and no blind area is generated.

[Brief description of the drawings]

FIG. 1 is an explanatory view of a method for manufacturing a cylindrical member of a mounting tool.

FIG. 2 is an explanatory view of a conventional mounting tool.

FIG. 3 is an explanatory diagram of surface tension acting on a sample with a mounting tool using an ultrafine metal wire.

FIG. 4 is a perspective view of a cylindrical member.

FIG. 5 is an overall view of a mounting tool.

FIG. 6 is a schematic diagram of an X-ray diffractometer using the sample mount of the present invention.

FIG. 7 is a view showing a mounted state of a crystal sample according to the embodiment of the present invention.

8A and 8B are diagrams illustrating an example of a mounted state of (a) a bipyramidal crystal and (b) a rectangular parallelepiped crystal.

[Explanation of symbols]

11: synthetic resin plate, 12: groove, 13: capillary tube,
15: Cutting tooth, 21: Rod, 22: Thin quartz plate, 23
... adhesive, 24 ... rod, 25 ... extra fine metal wire, 26
... adhesive, 27 ... protein crystal, 28 ... crystal mother liquor, 30 ...
Cylindrical member, 31: glass fiber, 32: epoxy resin, 34: pin, 35: magnet, 40: mounting tool, 50
... goniometer, 51 ... low-temperature gas blowing device, 52 ...
X-ray generator, 53: X-ray focusing mirror, 54: photoconductor,
60: Crystal sample

Claims (2)

[Claims] 1. A cylindrical member made of an amorphous material is fixed to the tip of a rod made of an amorphous material such that the axis of the cylindrical member is substantially perpendicular to the axis of the rod. Sample mount for X-ray analysis. 2. The sample mounting device for X-ray analysis according to claim 1, wherein the rod and the cylindrical member are made of fused quartz.