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Home > publications > IUCr Books > 50 years of x-ray diffraction of Crystallography on the occasion of the commemoration meeting in Munich, July
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Ewald and dedicated to the International Union of Crystallography on the occasion of the commemoration meeting in Munich, July , is now available on the IUCr web site. With contributions from many of the founding fathers of the the field, it remains after half a century a valuable and intriguing treasure-house of information and history. From Zurich, after the war, I visited von Laue who had not taken root in Frankfurt and had returned to Berlin.

He opened the door in person, a revolver in one hand and a dagger in the other. In the summer of , just before the outbreak of war, I had joined Sir Ernest Shackleton's expedition to the Antarctic as physicist, and sailed in the Endurance. The presence of the heavy element slightly modifies the diffraction intensities and the comparison of the diffraction pattern in the presence and absence of these heavy elements allows the estimation of the phases by triangulation, after having positioned the heavy atoms in the crystal lattice using methods known as Patterson functions [ 19 ].

The third method is anomalous dispersion , a specific property of the diffraction pattern when absorption of X-radiation is no longer negligible [ 20 , 21 ]. This method consists in varying the incident beam wavelength around the absorption edge of one of the atom type contained in the molecule.

Comparing the diffraction pattern at different wavelengths will allow the estimation of the phases using methods similar to that of the isomorphous replacement [ 22 ]. Selenium is often used because it has an absorption edge near to the wavelengths used e. For proteins, selenomethionine, an amino acid for which the sulfur is replaced by selenium, is generally introduced biosynthetically [ 23 ]. In the case of nucleic acids, modified bases containing bromine are frequently used [ 24 ].


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Once a first set of phases is estimated, a first electron density map is calculated. If this map is sufficiently interpretable, the macromolecule can be built step by step in this map Figure 8. A combination of automated algorithm and manual method available through interactive graphics softwares are used [ 25 ], leading to a final model composed of the three-dimensional coordinates of each atom of the cell content constituted by one or several macromolecules. From that first built model, the diffraction intensities are calculated by Fourier transform and compared to the intensities experimentally measured.

This comparison allows the step by step improvement of the model. This cyclical process is called the crystallographic refinement, alternating the search for global minimum of energy functions and manual reconstruction of the model [ 26 ]. The calculation of the electron density map left allows the building of the atomic model step by step middle and leads to the three-dimensional model of the structure right. The final step, downstream the structure determination by X-ray diffraction, concerns the interpretation of the structure and its integration into the biological context [ 27 — 29 ].

It consists in the understanding of the structural result as a three-dimensional object and the appreciation of its function at the cellular or evolution level. The description of the interatomic interactions, the secondary structures Figure 9 , the domains and their arrangement that defines the fold or the tertiary structure Figure 9 , as well as the characterization of the shape, the electrostatic properties and the quaternary structure based on the content of the cell in the crystal packing, are often complemented by the study of the macromolecule in solution, to better characterize its oligomeric Figure 9 and its dynamic behavior, alone or in the presence of interactors, if known.

In the case of enzymes, these studies will be coupled with enzymological approaches to determine the activity and the catalytic constants. A The protein structures are represented by three modes of representation see also Figure 2. B Protein structures are described in four levels, from primary to quaternary structure.

An analysis based on bioinformatics tools will allow to place the structure determined in the context of structural and evolutionary knowledge at a given time [ 31 ]. The lessons learned from these studies, often of primary importance, provide information including the classification of the structure and its sequence within a family counterparts, on the distribution and evolution of folding in the different domains of life viruses, bacteria, archaea, eukaryotes , on the possible function when it is unknown, on the catalytic site and its spatial conservation and sequence, on the degree of oligomerization or on the existence of interaction with other partners, proteins, nucleic acids or ligands.

A final type of study seeks to place the three-dimensional object into the context of the knowledge on the major biological mechanisms of live, such as knowledge on gene expression with transcriptomics, on complex formation with interactomics, etc … This information will include the characterization of the partners of the studied macromolecule at the scale of the cell or the whole organism.

All these steps, from the structure determination to the biological interpretation, far from being the end of the story, are often the beginnings of new structural studies Figure 3. These can be articulated around analyses of the relative importance of the components of the macromolecule, the aminoacids, by determining the structure of mutants, or the studies of the interactions with partners by determining the structure of macromolecular complexes. Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.

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Login to your personal dashboard for more detailed statistics on your publications. Edited by Alicia Esther Ares. We are IntechOpen, the world's leading publisher of Open Access books. Built by scientists, for scientists. Our readership spans scientists, professors, researchers, librarians, and students, as well as business professionals. Downloaded: Abstract Knowing the three-dimensional structure of biological macromolecules, such as proteins and DNA, is crucial for understanding the functioning of life.

Keywords biological macromolecules X-ray diffraction monocristal tridimensional structure. Steps upstream the structure determination The first step, a step that falls within biology and includes molecular biology and biochemistry techniques, is the production of highly pure macromolecule in large quantity. The diffraction data The crystals obtained during the previous step are fished using a small loop Figure 4 , cryo-cooled to protect them from radiation damage [ 10 ], and then placed into a monochromatic X-ray beam produced by an appropriate source, either a rotating anode generator available in crystallography laboratories or a synchrotron radiation, the latter producing significantly more intense beams [ 11 ].

From the diffraction data to the electron density Three main methods exist for the estimation of the phases [ 12 ]. From the electron density to the structural model Once a first set of phases is estimated, a first electron density map is calculated.

Fundamental Principles of Single-crystal X-ray Diffraction

The analysis of these diffraction data then allows the crystallographer to calculate the electron density, which is the distribution of the electron cloud of the macromolecule in the crystal. This electron density provided it is sufficiently precise—this preciseness depends on the resolution of the diffraction data—allows the localization of each atom of the molecule, and thus the determination of its coordinates in the three-dimensional space [ 6 ]. The principle of crystallography. A A monochromatic X-ray beam bombards a crystal frozen in a cryo-loop that rotates on itself.

The observed diffraction spots are the result of the impact on the detector of the wave diffracted by the electrons in the crystal. B Electron density map of a fragment of a macromolecule is represented left.

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The three-dimensional structure of a macromolecule here a protein is represented in three ways: all-atoms, backbone and cartoon representation see Figure 9. To get this three-dimensional structure, several steps that falls within multiple disciplines are required Figure 3. Each of these steps represents potential bottlenecks that need to be overcome. These are the production and the purification of the macromolecule, its crystallization, diffraction data collection and processing.

Another crucial step is the determination of the phases of the measured signal, absolutely required to calculate the electron density. The last step is the refinement of the built structure, called the model, which will then be interpreted in the context of its biological function. The analysis of the model will thus raise new questions leading to the resolution of other crystal structures, such as structure of a complex between the studied protein and its partners [ 7 ]. We will in the following sections describe each of these steps. The main steps of the three-dimensional structure determination of biological macromolecules by crystallography.

The first step, a step that falls within biology and includes molecular biology and biochemistry techniques, is the production of highly pure macromolecule in large quantity. Once the sequence of the macromolecule to be studied has been identified and characterized by bioinformatics analyses, the sequence corresponding to the gene of the macromolecule is cloned in an expression vector and produced classically in a bacterial organism typically Escherichia coli.

The macromolecule is then extracted from the bacterial cells and purified using chromatographic techniques. The next bottleneck is based on physical chemistry, specifically crystallization which addresses concepts such as solubility of molecules and their transition from soluble state to a solid crystalline ordered state [ 8 ].

This step, built on statistical screenings plays with the variation of parameters such as temperature, pH, concentrations of biological macromolecules, as well as nature and concentration of crystallizing agents and various additives [ 9 ]. Obtaining a single homogeneous crystal, that result to high quality diffraction data, represents a crucial step in the process of determining a macromolecular structure. In order to increase the success rate, crystallization robots are used today to screen more than several thousands of parameters. The size from tens to hundreds of microns and the morphology of the crystals are highly variable Figure 4 and are not necessarily related to their diffracting power and quality.

Crystals of biological macromolecules. Left, a typical crystallization plate used in crystallization robots that allows to screen 96 crystallization conditions.

Middle, different crystals of macromolecule. Right, the crystal is shown in its cryo-loop see Section 2. The black bar is microns.


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  • The crystals obtained during the previous step are fished using a small loop Figure 4 , cryo-cooled to protect them from radiation damage [ 10 ], and then placed into a monochromatic X-ray beam produced by an appropriate source, either a rotating anode generator available in crystallography laboratories or a synchrotron radiation, the latter producing significantly more intense beams [ 11 ].

    Under these conditions, the waves scattered by the electrons of the macromolecules that are three-dimensionally ordered in the crystal add up in given directions the diffracted beam is characterized by a structure factor, Figure 7 and generate a diffraction spot on the screen of the detector Figure 5A. All the spots, regularly spaced, form the diffraction pattern Figure 5A.

    This diffraction pattern is reconstituted by using several hundreds of images, each corresponding to an orientation of the crystal that rotates on itself during the measurement of the diffraction data Figure 2 and Figure 5B. The information contained in each diffraction spot is characterized by the amplitude and the phase of the structure factor characterizing the corresponding scattered wave. The three-dimensional distribution of the spots is directly related to the cell parameters, e. The distribution of the spot intensities is directly related to the electron density distribution the macromolecules in the cell.

    Mathematically, this means that the diffraction pattern is the Fourier transform of the electron density Figure 7. A The diffraction pattern or Fourier transform of a crystallized molecule generates a three-dimensional spot lattice bottom , whose background image corresponds to the Fourier transform of a single molecule top. B Example of detector image constituting the diffraction pattern. Hundreds of images are usually recorded. The spots at the image edge are high resolution spots, providing the most detailed information.