JP3439571B2 - Sample mount for X-ray analysis - Google Patents

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 particularly to a sample for X-ray analysis suitable for performing crystallographic structure analysis of biopolymer crystals at a low temperature of about liquid nitrogen temperature. Regarding mounts.

[0002]

2. Description of the Related Art Protein X-ray crystal structure analysis methods contribute variously to the elucidation of the three-dimensional structure, construction principle and reaction mechanism of protein molecules that are responsible for life-sustaining chemical reactions in vivo, and their importance will continue in the future. It is expected to increase more and more. However, a problem that is always troublesome for X-ray diffraction experiments of protein crystals is deterioration of crystallinity due to radiation damage of protein crystals. The details of the radiation damage mechanism of protein crystals have not been fully clarified, but the degree of radiation damage may have a strong correlation with the total X-ray dose to the protein crystals and the dose per unit time. Are known. It is considered that the X-rays absorbed in the protein crystal are converted into radical generation energy and heat, which eliminate the protein-protein interaction in the crystal, resulting in deterioration of crystallinity and dissolution. . For example, 12ke
0 at a rate of X-ray is 10 ¹⁰ / sec with the energy of the V.
When incident on a crystal with a linear absorption coefficient of 19 mm ^-1 ,
If all the absorbed X-rays are converted into heat, the temperature rise of the crystal is estimated to be about 1 mK / sec.

As a method for suppressing radiation damage, X-rays of short wavelength (0.03), which are unlikely to cause protein crystals to absorb, are used.
There are two conceivable methods: a method in which the generated heat is rapidly removed and a radical is suppressed by conducting a diffraction experiment at a low temperature. Since the former requires the use of a large-scale synchrotron radiation experimental facility, considering the measurement at the laboratory level, a diffraction experiment at a low temperature is desired and has been actually attempted.

The biggest difficulty in conducting a diffraction experiment at low temperature is that the water contained in a large amount in protein crystals precipitates as hexagonal ice. Ice precipitation destroys protein crystals from the inside. To prevent ice precipitation in the cooling process,
Replace the crystal mother liquor of the protein crystals with one containing an antifreeze agent,
Further, in the cooling, it is indispensable to expose the protein crystals directly to the cryogen for rapid and efficient cooling. In consideration of such requirements, the capillary tube that has been traditionally used in the conventional protein X-ray crystal structure analysis experiment cannot expose the protein mounted therein to the cryogen directly, and therefore, at low temperature. It is unsuitable as a crystal mount tool when conducting an X-ray diffraction experiment.

Mau suitable for rapid cooling of protein crystals
A thin quartz plate as shown in Fig. 2 (a) is used as the component.
There are known mounts that were used (Acta Cryst.B45, 190
-199 (1989)]. This mount is the tip of the quartz rod 21.
Thickness of about 100μm at the end, surface area of about 500 × 500μm²
Assembled by fixing the thin quartz plate 22 of
The protein crystal in the crystal mother liquor is a thin quartz plate 2
Mount on the surface of No. 2. In addition, FIG.
As shown in, platinum, tungsten with a diameter of about 80 μm,
Loop the ultrafine metal wire 25 made of constantan etc.
And bend it into a solid shape and fix it to the tip of the quartz rod 24 with an adhesive 26.
A fixed mount is also known [J. Appl. Cryst.2
3, 387-391 (1990)]. This mount is designed to
Using surface tension, the crystal is attached to the ultrafine metal wire 25.
It is something to be undone. The mounting tool shown in Figure 2
Also, the lower ends of the rods 21 and 24 are the same as those in the X-ray diffractometer.
It is used by fixing it to the ometer.

[0006]

By the way, in the mount using the thin quartz plate, slight irregularities on the surface of the thin quartz plate 22 cause mechanical microdeformation in the protein crystal, and the obtained X-ray diffraction data is obtained. May impair reliability. Further, the operation of scooping the crystal in the crystal mother liquor and placing it on a thin quartz plate and the subsequent operation of removing the crystal mother liquor are required, and there is a drawback that the handling is not quick.

Even in a mounting tool using an ultrafine metal wire, as shown in the schematic cross-sectional view of FIG. 3, as indicated by an arrow on the crystal 27 due to the surface tension of the crystal mother liquor 28 existing in the gap between the wire 25 and the protein crystal 27. There is a case that the correct diffraction data cannot be obtained because the crystal lattice is distorted due to a strong force. Further, a more serious problem is that since a metal wire is used, a so-called blind region is generated, and when the X-ray hits a loop made of the metal wire, a strong diffraction scattering pattern generated from the X-ray enters the detector, It is virtually impossible to measure the intensity of the reciprocal space region existing in such an arrangement. Further, since the loop wire has a circular cross section, there is a problem that it is difficult to set the crystal even if the protein crystal is sufficiently strong.

An object of the present invention is to provide a new crystal mount for low temperature X-ray diffraction which solves the problems of the prior art.

[0009]

[Means for Solving the Problems] The sample mounting tool used for low-temperature X-ray structural analysis of protein crystals and the like is (1)
High sample cooling efficiency, (2) No mechanical damage to the mounted sample crystal, (3) sample can be mounted by simple operation, (4) strong diffraction line from the mounting tool itself It is desired that the following conditions are satisfied: (5) the crystal can be oriented, (6) the coefficient of thermal expansion is small, and it is not destroyed by rapid cooling, and (7) X-ray transmission is possible. .

In order to satisfy the above condition, X according to the present invention
The sample mount 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 orthogonal to the axis of the rod. And The rod and the tubular member can be made of fused quartz or soda glass. The tubular member can be produced by, for example, cutting a fused silica capillary tube into slices. Further, the tubular member may be a cylindrical body having a circular cross section perpendicular to its axis,
It may be a tubular body having another shape whose cross section perpendicular to the axis is a triangle or a quadrangle.

[0011]

[Function] Since the portion for mounting a sample such as a protein crystal is cylindrical and the sample is exposed, the cryogen can be brought into direct contact with the sample for quick freezing. Since the sample adheres to the relatively wide and flat wall surface of the tubular member, the crystal can be mounted without distortion. The sample can be mounted by pivoting it on the wall surface of the cylindrical member by a simple operation of dipping the tip of the mounting tool in the sample solution and scooping out the crystal sample in the crystal mother liquor, and then removing the solution. No special operation required. That is, because the crystal sample in the crystal mother liquor is scooped into the internal space of the tubular member, the crystal plane of the crystal easily coincides with the surface created by the surface tension of the crystal mother liquor with respect to the tubular member,
By trying the scooping operation several times, it is possible to easily mount the crystal axis in a predetermined direction. For example, in the case of a bipyramidal crystal that is often found in protein crystals, as shown in FIG. 8 (a), it is easy to mount with two vertices positioned in a tubular member, and in the case of a rectangular parallelepiped crystal Is easily mounted with one of the crystal faces in close contact with the wall surface of the tubular member as shown in FIG.

Since the cylindrical member for mounting the sample is made of an amorphous material such as fused quartz or soda glass, strong diffracted rays are not generated from the mounting tool itself. Further, fused quartz and soda glass do not break even if they are brought into contact with a cryogen and rapidly cooled, and have sufficient X-ray transmittance.
Table 1 below shows a comparison between the performance of the sample mount according to the present invention and the performance of the sample mount using the conventional thin quartz plate or the ultrafine metal wire. Table 1 Sample mounting tool The present invention Thin quartz plate Ultrafine metal wire ───────────────────────────────────── Material Fused quartz Fused quartz Metal thin wire Cooling efficiency Good Good Good Mechanical load on crystal No Yes Yes Blind No No Yes Vertical axis Yes Yes No In this way, the sample mount for X-ray analysis according to the present invention is used for samples such as protein crystals. All the conditions required for low-temperature X-ray structural analysis are satisfied, and by using this sample mount, highly reliable X-ray diffraction data can be easily obtained.

Further, according to the protein X-ray crystal structure analysis using the sample mount for X-ray analysis according to the present invention, it is possible to carry out the structure analysis of the intermediate reaction state of the protein at a low temperature,
It can also contribute to the elucidation of problems such as how the thermal vibration of the protein itself and the dynamics of water molecules surrounding it correlate with the function of the protein.

[0014]

The present invention will be described in detail below with reference to examples. First, a method of manufacturing the sample mount tool 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 the groove has an outer diameter of 200 to 1000.
A fused silica capillary tube 13 having a thickness of 10 μm and a thickness of 10 μm was inserted and fixed. After that, as shown in FIG. 1B, a cutting tooth 15 similar to that used for cutting a chip from a wafer in the field of manufacturing semiconductor parts is fed at a feed speed of 0.5 mm / sec to a synthetic resin plate. A cut was made on the surface of 11 and the capillary tube 13 was cut at a plurality of points. Thus, as shown in FIG. 4, the outer diameter is about 700 μm,
A fused silica cylindrical member 30 having a height of 100 to 200 μm and a wall thickness of about 10 μm was produced.

As shown in FIG. 5, the produced cylindrical member 30.
Was adhered to the tip of the glass fiber 31 using epoxy resin 32. This epoxy resin retains its adhesive performance even in rapid freezing with 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 portion of the X-ray diffraction apparatus so that the irradiation X-rays pass through the position of the cylindrical member 30. The mounting tool 40 manufactured in this manner is further adhered to a metal pin 34 such as brass for use. 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 the pin 34 can be easily positioned during rapid cooling with low temperature nitrogen gas.

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

The measuring method using the X-ray diffractometer of FIG. 6 will be described below. The difficulty in collecting data on protein crystals at low temperatures is that water, which occupies most of the solvent region of protein crystals, is frozen as hexagonal ice upon cooling. In order to prevent the formation of hexagonal ice in the initial cooling and freeze the water into a glassy state, it is necessary to penetrate the crystal mother liquor with a cryoprotectant and increase the cooling rate of the crystals. The water molecules in the protein are frozen into a glass by the combined use of an antifreezing agent and quick freezing. Antifreeze agents have the effect of lowering the equilibrium freezing point, suppressing the uniform growth of ice, and increasing the supercooling phenomenon with respect to the phase transition of water. Methanol, glycerol, 2-methyl-2,4-pentanediol, saccharose, polyethylene glycol and the like can be used as the antifreezing agent that permeates the protein crystals. 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)], a low-density packed beef trypsin orthorhombic crystal was prepared. The crystal mother liquor is 2.1 M ammonium sulfate, 1% (w / v) benzamidine, 1 mM calcium chloride, 20 mM sodium cacodylate (pH
6.0). 25% (w /
v) Sucrose was added, and the solvent replacement by dialysis was performed to 12
I went on time. Then, the trypsin crystal sample was mounted on the mounting tool 40 shown in FIG. 5 and flash frozen with 98K low temperature nitrogen gas.

The trypsin crystal sample is 250 × 150 × 4
The cylindrical member 3 of the mount having a size of 00 μm ³ and with the crystal axis oriented in the vertical direction as schematically shown in FIG.
It was frozen and mounted at 0. The mount on which the crystal sample was mounted was mounted on a goniometer 50 of an X-ray diffractometer as shown in FIG. 6, and low temperature nitrogen gas was blown from a low temperature gas blowing device 51 to maintain the temperature at 100K. X-ray the CuKα ray (wavelength 0.14518 nm) from the X-ray generator 52 operated at 50 kV and 80 mA.
The line collecting mirror 53 collects the light, and the cylindrical member 30 of the mount tool
The trypsin crystal sample mounted on the plate was irradiated and measured by the vibrating crystal method. The measurement results are shown in Table 2. Table 2 Lattice constant a = 6.33 b = 6.26 c = 6.80 nm Resolution 4.6-0.18 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 formula (1), and is an amount serving as an index indicating how accurate the measurement is.

R _merge ^I = Σ _h Σ _i | I _i (h)-<I (h)> | / Σ _h Σ _i I _i (h) (1) where h is the index of the reciprocal lattice point (3 Dimension vector), and I (h) is the reflection intensity (integrated intensity) of the index h. In the measurement, the intensity of a desired h group (usually about 10,000 pieces) is measured, but in the vibrating crystal method, a plurality of desired I (h) groups can be measured. Assuming that we can measure n I (h) now, write it as follows, and calculate the average <I (h)>
And

I ₁ (h), I ₂ (h), ..., I _n (h) Therefore, 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 at all the measurement points. As can be seen from the above definition, R _merge ^I indicates that the smaller the value, the higher the statistical accuracy, and it is zero in an ideal experiment, but 0.06 in a normal experiment at room temperature in a laboratory.
Values of 0.07% to 0.07%. Although the size of the crystal is in a relatively small size in view of collecting data in a laboratory, a low temperature experiment using the mounting tool of the present example allows sufficient exposure time and radiation damage. Since it is not present, R _merge ^I shows a good value of 0.049.

According to another experiment conducted by the present inventors, a crystal whose data can be collected only at a resolution of 0.6 nm due to radiation damage in the measurement at room temperature was subjected to a low temperature experiment using the crystal mount of the present invention. The resolution could be greatly expanded to 0.25 nm. Quick 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 foaming property and thus has a slow cooling rate. Further, once the cooling is completed, the measurement is performed while constantly cooling with a low-temperature nitrogen gas spraying device so that the temperature of the liquid nitrogen becomes 2
It is possible to collect diffraction intensity data at any temperature up to about 20K.

The above-described mount tool, which is produced by cutting a capillary tube, which is usually used for experiments at room temperature, into a thin ring shape, can transmit X-rays in all directions and uses its wall surface. This has the advantage that the crystal axis of the crystal can be oriented with respect to the incident X-ray beam. A technique for collecting diffraction intensity data for protein X-ray crystal structure analysis at low temperature is used both in the laboratory and in experiments using synchrotron radiation X-rays.
It is important in reducing the radiation damage of protein crystals due to X-rays. This technique allows long-term exposure of biopolymer crystals to X-rays, resulting in statistically reliable diffraction intensity data. Furthermore, by applying this technique, it becomes possible to investigate the physical properties of proteins such as the temperature dependence of internal vibration of proteins and the role of water molecules bound to the proteins.

[0024]

According to the X-ray sample mount of the present invention,
A sample such as a protein crystal can be vertically mounted without being stressed. In addition, since a strong diffraction line is not generated from the mount itself and a blind area is not generated, clear diffraction data can be obtained.

[Brief description of drawings]

FIG. 1 is an explanatory view of a method for manufacturing a tubular member of a mount tool.

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

FIG. 3 is an explanatory diagram of surface tension acting on a sample by a mount 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 view of an X-ray diffractometer using the sample mount of the present invention.

FIG. 7 is a diagram showing a mounted state of a crystal sample in an example of the present invention.

FIG. 8 is a diagram illustrating an example of a mounted state of (a) a bipyramidal crystal and (b) a rectangular parallelepiped crystal.

[Explanation of reference numerals] 11 ... Synthetic resin plate, 12 ... Groove, 13 ... Capillary tube,
15 ... Cutting teeth, 21 ... Rod, 22 ... Thin quartz plate, 23
… Adhesive, 24… Rod, 25… Extra fine metal wire, 26
… Adhesive, 27… Protein crystals, 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

─────────────────────────────────────────────────── ─── Continuation of the front page (56) Reference JP-A-5-164737 (JP, A) Actual development Sho 61-67548 (JP, U) (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 23/00-23/227 G01N 1/00-1/44

Claims (3)

(57) [Claims] 1. A Rod X-ray analytical sample mounting device, characterized in that the tubular member to the tip axis of the cylindrical member is fixed so as to be substantially perpendicular to the axis of said rod. 2. The rod and the tubular member are amorphous materials
The sample for X-ray analysis according to claim 1, characterized in that
Mount fixture. 3. The sample mount for X-ray analysis according to claim 1, wherein the rod and the tubular member are made of fused silica.