X-ray crystallography[1]It is a subject that uses X-ray to study the arrangement of atoms in crystals.More precisely, the electronic pairX-rayX ray crystallography can obtain the distribution of electron density in the crystal, and then analyze it to obtain the position information of atoms, that iscrystal structure。
It can provide reliable answers to many questions related to the complementary structure, such as the overall folding, ligand or substrate binding, and the specific information of the interacting atoms.Applied X-ray crystallography[1]The structure and function of macromolecules such as protein and DNA can be understood.Accurate information of molecular structure is a prerequisite for rational drug design and structure based functional research.
Since all atoms contain electrons, andX-rayOfwavelengthRange: 0.001 － 10nanometer(i.e. 0.01-100Angstrom）Its wavelength is comparable to the distance between bonding atoms (around 1-2 angstroms), so X-ray can be used to study the structure of various molecules.However, up to now, it is not possible to use X-ray to image a single molecule, because there is no X-raylensX-ray can be focused, and X-ray candiffractionThe ability is very weak and cannot be detected.The crystal (generallysingle crystal）It contains a large number of molecules with the same orientation. The superposition of X-ray diffraction on these molecules can generate enough signals to be detected.In this sense, a crystal is an X-ray signal amplifier.X-ray crystallography combines X-ray withCrystallographyLinked together so that various types ofcrystal structureConduct research, especiallyproteinCrystal structure.
Crystallography can provide reliable answers to many questions related to complementary structures, such as overall folding, ligand or substrate binding, and specific information about the atoms acting on them.Applied X-ray crystallography[1]The structure and function of macromolecules such as protein and DNA can be understood.Accurate information of molecular structure is a prerequisite for rational drug design and structure based functional research.
Diffraction data → data processing → phase analysis → modeling → model correction → model verification
⑤ Understand the relationship between structure and function
crystal growth
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Protein crystal under microscope
principle：
Protein crystal[3]The internal structure is a three-dimensional space periodic, orderly and repeated arrangement, which requires that the chemical composition and molecular conformation of each crystal repeating unit (molecule or its complex) are uniform.
method：
In order to obtain diffractive single crystals, it is necessary to grow the purified biological samples.There are many methods for crystal growth, such as gas phase diffusion, liquid phase diffusion, temperature gradient, vacuum sublimation, convection and so on. At present, the most widely used method for crystal growth is gas phase diffusion.Gas phase diffusion method can also be divided into hanging drop method, sitting drop method, Sanming treatment method, oil drop method and micro dialysis method.Among them, the hanging drop method is the most frequently used.
(The above methods are chemical methods. Generally, researchCondensed matterPhysicsThe zone melting method is most commonly used, which is based on polycrystalline materials and partially melted and recrystallized by applying high temperature locally, so as to gradually obtain large crystals. Polymer materials usually cannot withstand excessive temperatures, so this method cannot be used)
After obtaining the initial crystal growth conditions, it is often necessary to optimize the crystal growth conditions, including adjusting the precipitator concentration, pH value, sample concentration, temperature, ionic strength, etc.
Diffraction data collection
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After obtaining the single crystal, it is necessary to carry out diffraction experiments, that is, hit the crystal with X-ray to generate diffraction and record the diffraction data.There are two main sources of X-ray. One is used on the commonly used X-ray instrument, which bombards the copper target (or molybdenum target) with high-energy electron flow to generate X-ray with multiple characteristic wavelengths. The wavelength of CuK α used is 1.5418Å;The other is to usesynchrotron radiation[4]The wavelength of the generated X-ray can be changed.Synchrotron radiation X-rays can be divided into angular dispersive synchrotron radiation (ADXD) and energy dispersive synchrotron radiation (EDXRD). The experimental principle of angular dispersive synchrotron radiation is the same as that of ordinary X-ray diffractometer, but the wavelength is lower (such as 0.6199Å) and the energy is higher;The white light incident is used for energy dispersion, that is, the incident light has a continuous wavelength, and the collected diffraction signal is conducted at a fixed angle. Its resolution is lower than angular dispersion synchrotron radiation, and its technical requirements are also lower.Now the Beijing Synchrotron Radiation Experimental Station (BSRF) in China has been upgraded to angular dispersion.
Diffraction data (including the position and intensity of diffraction points) are recorded by image plate orCCDDetector.
Data analysis
By analyzing the recorded diffraction data, we can obtain the crystal system and the corresponding bragg lattice, as well as the miller index and the corresponding intensity of each diffraction point in the reciprocal space.
Indexation
Intensity integration, combination and amplitude reduction
Discussion on some problems of crystallographic parameter measurement
Common data collection and processing procedures
Protein crystal[5]Evaluation of diffraction intensity data quality
Crystal structure analysis
Because the crystal diffraction is actually the superposition of the electron density of each atom in the crystal to the X-ray diffraction, the diffraction data reflects the result of the Fourier transform of the electron density, which is expressed by the structure factor.The distribution of electron density in the crystal can be obtained by inverse Fourier transform of the structure factor.The structure factor is related to the wave equation. To calculate the structure factor, we need to obtain three parameters in the wave equation, namely the amplitude, frequency and phase of the wave.The amplitude can be directly calculated from the intensity of each diffraction point, and the frequency is also known, but the phase cannot be directly obtained from the diffraction data, so the "phase problem" in crystal structure analysis arises.
The direct method and Patterson method are used to solve the phase problem in crystal structure analysis.The main methods for analyzing the structure of biological macromolecules include molecular replacement, isomorphic replacement and abnormal scattering.
Determine phase
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Multiple isomorphic replacement (MIR)
Heavy metal atoms with high X-ray scattering ability are used as identification atoms.This kind of macromolecule replaced with heavy atoms should have the same cell parameters and space group as the original crystal without heavy atoms, and most atoms have the same position, so it is called isomorphic replacement.From the diffraction data of these crystals containing heavy atoms[6]The position of heavy atoms can be solved by the method ofStructural factorAnd the phase angle, and then calculate the phase angle of the original crystal without heavy atoms by using the phase angle relationship, and solve the structure.More than one kind of heavy atom is often used for replacement to obtain several isomorphic replacement derivatives, which is called multi pair isomorphic replacement method.
Multi wavelength anomalous scattering (MAD)
There is a Friedel's law in crystal diffraction, that is, no matter whether there is a symmetry center in the crystaldiffractionThere is always a symmetric center in, that is, FHKL=FHKL.However, when the X-ray wavelength used is close to the absorption edge of an element in the sample to be measured, the above law is not obeyed, that is, FHKL ≠ FHKL.This is due to the abnormal scattering of electrons[7]This phenomenon can solve the phase angle problem of the object to be measured.In general, this method is often associated with heavy atomsIsomorphous replacementMethod.After receiving the diffraction data of isomorphic substituents, change the wavelength of the incoming ray to the absorption edge near the heavy atom, and collect the data again. There is abnormal scattering in this set of data, so we can use these two sets of data to calculate the phase.For example, multi isomorphic replacement method, if several sets of abnormal scattering data are collected for different elements with several different wavelengths of X-ray, more correct and complete phase information can be obtained, which is the multi wavelength abnormal diffraction method (MAD).
Both of them have the same point: they use the properties of heavy atoms to solve the phase angle problem.
Difference between the two: MAD is based on MIR and uses multiple wavelengths to complete the required information.
Molecular modeling and structure modification
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protein structure[8]Difference ofSpatial scaleAnalysis requires different equipment
protein
Protein complex
Cells
organization
individual
ten-9m
ten-8m
ten-5m
ten-2m
ten-1m
X-ray, NMR, MS
X-ray, nuclear magnetic resonance, mass spectrometry, electron microscope
Optical microscope, electron microscope, soft X-ray spectrum
Optical microscope, electron microscope, infrared spectrum
Magnetic resonance imaging MRI, electron emission tomography PET, single photon emission tomography SPECT
