Crystallography and Lectin Structure Database - CNRS

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16 U. Krengel and A. Imberty the first Nobel Prize in physics for his discovery in 1901. A few years later, it was proposed that the wavelength of X-rays were of the same magnitude as interatomic distances, and in 1912, a crystal diffraction experiment confirmed this hypothesis. The first crystal structure was solved in the same year. It then took a few decades until this method could be successfully applied to large and fragile macromolecules such as proteins and DNA. Dorothy Crowfoot Hodgkin played a leading role in this work. The year 1962 marked a period of particular recognition of macromolecular X-ray structure analysis, as both the Nobel Prize in physiology and that in chemistry went to landmark X-ray structures: Watson, Crick, and Wilkins received honors for their famous 3D-structural model of DNA and Kendrew and Perutz for the first protein structures solved, those of myoglobin and hemoglobin. As the name “X-ray crystallography” suggests, protein crystals are a necessary precondition for the study of proteins or protein ligand complexes by this method. Crystals are needed in order to enhance the scattering power of the subjects under investigation. Normally, molecules scatter X-rays only weakly, but if they are regularly arranged in a crystal (Fig. 1), scattering by one molecule is reinforced by all the other molecules in the crystal and an X-ray diffraction pattern can be recorded. Crystallizing a protein is not that difficult in pure technical terms (see Section 5 and the book by Bergfors [2] for practical instructions), but in practice, the path to well-diffracting protein crystals is often long and thorny. In fact, protein crystallization is one of the two main bottlenecks of X-ray crystallography. Practically, a good advice is to start out with ultra-pure protein. This will in many cases significantly increase the chances for success. Figure 1. Protein crystals and crystal architecture. Crystals are three-dimensional macroscopic objects that are constructed from smaller units, so-called “unit cells,” by translation along the unit cell axes. The unit cells in turn contain even smaller units termed “asymmetric units,” which are related to each other by crystallographic symmetry, such as the rotation about a symmetry axis. The crystal pictures were kindly provided by Åsa Holmner Rocklöv and the picture of the crystal architecture was reprinted with permission from [5].
Crystallography and Lectin Structure Database 17 The second major bottleneck in X-ray crystallography is to overcome the socalled Phase Problem, the problem that only the amplitudes but not the phases of the diffracted X-rays can be obtained from conventional X-ray diffraction experiments. This problem is all the more serious as the phases contain much more important information than the amplitudes. For macromolecular structure analysis, there are mainly two methods to circumvent the phase problem: Molecular replacement (MR) and heavy-atom phasing methods. The first method is relatively simple. It involves placing a similar molecule in the lattice of the crystal under investigation (in the same orientation and position as the target molecule) and then calculating the phases from this model by means of a Fourier analysis. This method is quick and works rather well if the similarity between target and search model is significant and the crystal symmetry not too high. The method, however, can only be applied if a suitable search model exists (which happens increasingly often as more and more structures are solved and deposited in databases). If no such search model is available, there is usually no way around heavy-atom phasing methods, such as multiple isomorphous replacement (MIR). These techniques require the introduction of heavy atoms (such as mercury and lead) into the native crystals. Heavy atoms scatter X-rays particularly strongly (as they contain many electrons, which are the main scattering particles of the atoms) and leave traces in the X-ray diffraction pattern, from which initial phases can be derived. Once the relative phases are determined for all reflections (“reflections” being the proper scientific term 1 for the diffraction spots of a crystal diffraction pattern), the information from amplitudes and phases can be combined in a Fourier synthesis to yield a three-dimensional electron density map of the crystal structure, which can in turn be interpreted in terms of atomic coordinates (for an overview of an X-ray crystal structure analysis, see Fig. 2). The final steps of an X-ray structure analysis are crystallographic refinement and validation. During the refinement of the model, the differences between observed and calculated (model-derived) amplitudes are being minimized, in order to obtain a precise and accurate model of the crystal structure under investigation. A number of very good textbooks are available that cover protein crystallography in more detail, e.g. the books by Blow [3], Drenth [4], McPherson [5], and Rhodes [6]. 2.1. Recent technical advances in the field During the past two decades, many advances in the field of X-ray crystallography helped to significantly speed up the process from crystal to final structural model. Recombinant techniques [7–9] are now routinely employed to obtain large quantities 1 The term “reflection” refers to Bragg’s interpretation of X-ray diffraction in terms of reflection of the X-ray beam at crystal lattice planes. Constructive interference only occurs when Bragg’s law is fulfilled, meaning that the X-rays hit a certain set of lattice planes with spacing d at an angle , such that 2dsin.
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Crystallography and Lectin Structure Database - CNRS

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