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Method for preparing enzyme and substrate compound crystals

A complex and crystal technology is applied in the field of preparing enzyme and substrate complex crystals to achieve the effect of improving quality, high resolution and expanding the scope of application

Inactive Publication Date: 2013-04-24
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The key group in the mutant enzyme and the method of selecting substrate analogues have the disadvantage that the obtained structure is an approximate structure, and the low temperature technology is only used to improve the diffraction resolution, and does not involve the inhibition of enzyme activity.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Prepare a low-temperature crystal plate, moisten the paper strip containing glycerin with water, then fill the drip hole with water, and cover the glass cover to maintain the humidity of the environment.

[0034] 2. Prepare antifreeze: 1 μl 50% (v / v) glycerin, 2 μl pool solution (0.1M sodium 4-hydroxyethylpiperazineethanesulfonate, pH 7.5, 10% (v / v) isopropanol and 20% poly Ethylene glycol 4000 (w / v))

[0035] 3. Use a nylon ring to pick out the 6-phospho-β-glucosidase protein crystals, put them into antifreeze, and quickly place them in a -20°C low-temperature refrigerator for 10 minutes.

[0036] 4. Quickly take out the protein crystal plate, and quickly add 3 μl of 20 mM substrate p-nitrophenyl-β-D-glucopyranoside-6-phosphate.

[0037] 5. Put the crystal plate back into the -20°C low-temperature refrigerator again, and wait for 10 minutes.

[0038] 6. Take out the crystal plate quickly again, pick out the protein crystal and substrate complex with a nylon ring, ...

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PUM

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Abstract

The invention relates to a method for preparing enzyme and substrate compound crystals. The method comprises the following specific steps of: 1) placing enzyme protein crystals in an antifreezing solution, and placing at the temperature of -20 DEG C for 5 to 10 minutes; 2) quickly taking the enzyme protein crystals out, and adding substrate of which the volume is equal to that of the antifreezing solution; 3) placing the enzyme protein crystal and substrate compound at the temperature of -20 DEG C again for 5 to 10 minutes; and 4) taking out, quickly placing in liquid nitrogen and fixing. By the method, the real enzyme-substrate compound structure can be obtained; and the occupancy and the space stability of the substrate can be further improved, so that the quality of the electron density of the substrate is improved. On the basis that the space ordering of the biomacromolecule and compound crystals is improved, diffraction data with high resolution ratio can be obtained.

Description

technical field [0001] The invention relates to a method for preparing enzyme-substrate complex crystals. Background technique [0002] Accurately grasping the structure of biological macromolecules and their complexes is the basis for understanding life processes, and the analysis of their three-dimensional structures can give a lot of information about themselves and their systems. [0003] The method of determining the crystal structure of biological macromolecules and their complexes by using the X-ray diffraction system of biological macromolecules is the main method in the field of structural biology research. This work can be roughly divided into the following four links: preparation of high-purity macromolecular samples; crystal acquisition; structure analysis; biological research guided by structural information. Under the current technical conditions, the quality of the sample crystal is the main reason affecting its structure analysis. [0004] The main reason a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28
Inventor 李宏滨刘一苇
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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