Crystallography and Lectin Structure Database - CNRS

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26 U. Krengel and A. Imberty and characterize the binding site. There are, however, also cases were neither cocrystallization nor soaking give any results, either because the ligand does not bind strongly enough or because ligand binding is not compatible with crystal formation. (Tip: Binding can easily be checked by calculating difference Fourier maps of either Fo–Fo or Fo–Fc type for the obtained crystals.) If no electron density is visible for the carbohydrate ligand, the option remains to obtain further insight into ligand binding by modeling the compound into the combining site of the protein structure. Modeling the ligand into the binding site, with or without supporting electron density, requires a 3D structural model of the carbohydrate ligand. This can be obtained either from scratch (e.g. using the modeling tool “Sweet” from the http://www.glycosciences.de web site, which converts carbohydrate sequences into 3D models) or from a structure database (via the Uppsala HIC-Up server at http://xray.bmc.uu.se/hicup/) or directly from the PDB (http://www.pdb.org/) or other appropriate databases such as the Cambridge Structural Database (CSD, http://www.ccdc.cam.ac.uk/) or the Glyco3D server of CERMAV (http://www. cermav.cnrs.fr/glyco3d). The model of the carbohydrate ligand should then be refined and checked as carefully as the protein structure itself. Currently, however, tools for analyzing carbohydrate structures are not as widely known and applied as those for protein or DNA structures [50]. A good tip is to check out the web site at http://www.glycosciences.de. A couple of tools, such as pdb-care (to check carbohydrate residues in pdb files for errors [58]) or carp (which generates Ramachandran-like plots for carbohydrates [59]) or even GlyProt (to identify glycosylation sites in proteins and automatically attach them in silico) make life of structural glycobiologists significantly easier. Another database, currently under development, is EuroCarbDB (http://www.eurocarbdb.org/databases). And questions concerning carbohydrate nomenclature may be resolved by consulting the web site http://www.chem.qmul.ac.uk/iupac/2carb/. 2.4. Electron microscopy and neutron diffraction Two other methods for macromolecular structure determination exist that are very similar to X-ray crystallography. These are electron microscopy [60] and neutron diffraction [61]. Neutron diffraction is more of an oddity in structural studies. In this method, the obtained 3D-crystals are exposed to a neutron beam instead of X-rays. Neutrons, produced in a nuclear reactor, have no electrical charge, a mass almost the same as a proton, a nuclear spin of 1/2, and a magnetic moment. Thermalized fission neutrons (thermal neutrons) have as in the case of X-rays, wavelengths of the same order of magnitude as the diameter of atoms and are therefore well suited for the study of condensed matter. The interaction between neutrons and the atoms in the crystal lattice differs in several respects from the interaction between atoms and X-rays. The major difference is caused by the fact
Crystallography and Lectin Structure Database 27 that X-rays are diffracted by electron clouds, whereas neutrons are scattered by nuclei. The principal problem associated with neutron crystallography of proteins is caused by the low flux of neutrons through the sample. The interaction of neutrons with atoms in the biomaterial is therefore weak and the crystals have to be very large, generally at least 1 mm 3 in volume. In addition, the exposure times become very long, typically several weeks or more. The required large crystal size poses a serious limit to most structural studies of protein crystals with this method. In cases where such large crystals can be obtained, however, as a reward, hydrogen atoms in the structure can be resolved. Knowledge of the exact location of hydrogen atoms in a given macromolecule is crucial for the understanding of many biological problems, and this feature is probably the most important reason for using the neutron diffraction technique [62]. Electron microscopy has traditionally been used to study heavy-atom soaked tissue sections at relatively low resolution [63]. This method can, however, also be used to study the structures of macromolecules. This is done with two variations of the technique: electron crystallography [64] and single-particle cryoelectron microscopy [65–67]. Single-particle studies require relatively large molecule sizes (corresponding to molecular weights of at least 100–200 kDa). The resolution of the obtained structures is not particularly impressive (20–30 Å); nevertheless, single-particle studies are invaluable in cases where no crystals can be obtained. Even if only a rough molecular envelope can be deduced, this can provide starting phases for higher resolution X-ray crystal structures and, more importantly, available atomic-resolution models of macromolecule domains can be modeled into the molecular envelope of a complete protein complex to give a picture of the biological system in action [68–70]. In contrast to single-particle studies, electron crystallography requires crystals. This technique is analogous to X-ray crystallography, only that an accelerated electron beam is used instead of an X-ray beam, and 2D-crystals are used instead of 3D-crystals. Nevertheless, in order to obtain a three-dimensional image, the 2D-crystals need to be placed on a grid support and then tilted in the electron beam. Since usually the highest tilt angles obtainable are ca. 70, electron diffraction data are usually less complete than X-ray diffraction data. They are also much weaker. Electron crystallography, however, has one huge advantage over X-ray crystallography: it does not have a phase problem. Since electrons can be focused with magnetic lenses, a direct image of the structure can be obtained in addition to the electron diffraction pattern. Nevertheless, the diffraction pattern is valuable due to its superior data quality. As for X-ray diffraction, also electron diffraction gives a discontinuous diffraction pattern featuring spots (the reflections) on lattice planes. Between the reflections, the intensity should be zero, which can be taken as a condition to accurately estimate the background of the diffraction data and hence derive high-quality primary data. Electron crystallography is a method that is particularly suitable for macromolecules that readily form 2D-crystals. As such, this method is predestined for the
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