Crystallography and Lectin Structure Database - CNRS

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18 U. Krengel and A. Imberty Figure 2. Overview over an X-ray crystallographic analysis (from crystal to refined structural model). Steps required are (1) protein crystallization, (2) X-ray data collection, (3) phasing to obtain (4) a 3D-electron density map (by Fourier Synthesis), into which (5) a structural model can be built that needs to be (6) refined and validated before publication. The picture shows the real-life success story for the Erythrina crystagalli lectin [139] (picture kindly provided by Cecilia Cronet). of homogenous protein preparations, while traditionally, lectins were directly extracted from their natural sources. It should be stated here that there is nothing wrong with using the traditional methods, especially if the natural lectin sources are abundant, but work can benefit from the use of recombinant techniques in several different ways: Apart from the obvious advantage of reproducibly obtaining homogenous protein sample, molecular cloning opens the door to mutagenesis studies for functional investigations. But more than that, recombinant techniques may also be used to introduce unnatural amino acids like seleno-methionine (Se-Met) and seleno-cysteine (Se-Cys) [10–13] into the protein sequence. This allows, among others, the rational introduction of heavy atoms for phasing and thus avoids the often very time-consuming trial-and-error searches for suitable heavy-atom derivatives. In effect, biomodification significantly speeds up the structure determination process. These new labeling methods, however, can only take effect due to the development of a new phasing method, called multiple anomalous dispersion (MAD) [14]. This method, also requiring the introduction of heavy atoms, is similar to the traditional MIR method, but it makes use of the anomalous scattering of heavy atoms at the absorption edge. Instead of collecting several datasets of different isomorphous heavy-atom derivatives and comparing them with the X-ray data from the native, unmodified protein crystal, datasets are collected from, ideally, one (derivatized) crystal, at several different wavelengths. This method requires far
Crystallography and Lectin Structure Database 19 weaker heavy-atom scatterers and has become increasingly popular in recent years [15]. In some cases, the method works without any modification of the protein at all, e.g. if the protein naturally contains metal ions (as in Fe–S clusters); and there have even been reports of phasing based on the anomalous scattering of the native sulfurs in cysteine residues [16–18]. If combined with biomodification, i.e. Se-Met labeling, one may not only rapidly and rationally obtain an experimental electron density map, but in addition take advantage of the known sequence positions when building the model. Phasing based on Se-Met labeling can be done even for relatively large proteins, provided that the proteins contain enough methionine residues (1 per 100 amino acid residues). If the number of Se-Met residues is very high, traditional Patterson methods fail to identify the large number of peaks. However, the selenium substructures can be located by advanced direct method approaches [19]. These new approaches can even identify other kinds of substructures like sulfur or halide atoms or even missing sulfur atoms from radiation-induced decay [20, 21] and hence open the way to completely new phasing methods [22–24]. The MAD method relies on data collection at the absorption edge of the anomalous scatterers and this in turn requires the use of very specific wavelengths. For traditional X-ray sources, the wavelength is determined by the characteristics of the anode material, which is in general incompatible with the wavelength(s) needed for a MAD experiment. In order for the MAD method to become generally applicable, it was therefore essential that a tunable X-ray source was available. These requirements are exquisitely met by synchrotrons. Originally developed for particle physicists, synchrotrons with time became one of the most important tools for protein crystallographers [25–29]. Nowadays, every synchrotron has its dedicated protein crystallography beamline(s). Main advantage, apart from the tunable wavelength, is the high brilliance of the X-ray beam, a nearly parallel beam of very high intensity, which allows data collection even for tiny and poorly diffracting crystals, to the limit of their diffraction power. In recent years, X-ray data for the great majority of reported crystal structures has been collected at synchrotrons. Interestingly, this trend could soon start to reverse, as also the home sources are becoming more and more powerful, especially those equipped with advanced micro-focusing optics. Even table-top synchrotrons are now appearing on the market [30]. A high intensity X-ray beam, however, also comes at a price: the higher the intensity, the more serious is the radiation damage that the crystals have to withstand. As a result, crystals quickly loose diffraction power when exposed to synchrotron radiation. They “die” in the X-ray beam. Their lifetime can, however, be extended, if the experiments are performed at low temperature. Around the beginning of the 1990s, cryotechniques were developed [31–33], which employ low-molecular weight organic compounds (glycerol, ethylene glycol, sugars, etc.) or oils as cryoprotectants to prevent the formation of ice crystals that would destroy the crystal. If transferred quickly (“flash-freezing”) into liquid nitrogen or propane, the aqueous solvent instead freezes like a glass, allowing high-quality data to be collected for extended
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