Crystallography and Lectin Structure Database - CNRS

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40 U. Krengel and A. Imberty have been developed based on successful crystallization reports. These screens provide for an easy start. In general, it is recommended to start with the most common screen, Screen nr. 1, and then continue with other screens based on the obtained results. It may, however, also make sense to take the characteristics of the protein you are trying to crystallize into consideration from the very beginning. Are you working with a membrane protein? Then, you should use a screen developed for membrane proteins. Are you working with a lectin? Then, you may check out the information compiled in the BMCD at http://wwwbmcd.nist. gov:8080/bmcd/bmcd.html (searching by keyword for “lectin”). Is there a common theme? In this case, you might like to devise your own crystallization screen. Maybe, one of the proteins in the database (either the crystallization database or the PDB) has especially high sequence identity to the protein you interested in? Then, the crystallization conditions for that particular protein may pave your way to success – best of course if you combine the two approaches and try out commercial sparse matrix screens as well as your own tailor-made ones! If you are in the lucky situation that you have a dynamic or static light scattering device at hand, it can be a good idea to test how the protein behaves under different conditions (temperatures, buffers, etc.) before setting up the first crystallization experiments [121, 122]. Especially the temperature can be well worth testing. Conditions that indicate a monodisperse solution are much more likely to yield crystals than polydisperse solutions that contain various degrees of protein aggregates. The next decision to take, concerns the crystallization method. Some techniques lend themselves better to screening then others. Very suitable are methods for which multi-well plates are available (either 96-well or 24-well plates). The plates do not have to be special-made for crystallization; just usual tissue culture plates will do. Personally, we prefer to start with the hanging-drop technique shown in Fig. 8. The setups are really easy to prepare: (1) Pipette 0.5–1ml of the screening solution into the reservoir well (make sure that it is well-mixed in case of hand-made viscous solutions like PEG mixtures). (2) Grease the rim of the wells with either silicon oil (e.g. NVH oil from Hampton Research) or vacuum grease. (3) Place a clean cover slip (usually silanized) beside the plate (e.g. on the plate’s lid, which provides a convenient and clean surface). (4) Pipette first the protein solution (1–2l) onto the cover slip and then add the reservoir solution on top of it (usually the same volume) (DO NOT mix the solutions by pipetting up and down, because this can lead to protein denaturation). (5) Take the cover slip and place it upside-down onto the well with the reservoir solution.
Crystallography and Lectin Structure Database 41 Figure 8. Protein crystallization by the hanging-drop vapor-diffusion technique. The protein-containing drop is placed on a cover slip and hung upside down over a container with reservoir solution (left). Upon equilibration, the drop shrinks and the concentration in the drop rises until it matches the vapor pressure from the reservoir. If the conditions are just right, protein crystals will appear in the drop after some time (middle). Crystallization follows the phase diagram shown on the right, from low concentrations of protein and precipitating agent (undersaturation) over the solubility line into the supersaturated state. Crystal nuclei are formed at somewhat higher concentration than optimal for crystal growth. Upon crystallization, the protein concentration in the drop will again decrease until it reaches the solubility line. In an alternative to spontaneous crystallization (shown here), seeding techniques may be used, in which tiny crystal seeds are placed directly into the metastable zone. This picture was prepared by Alexandre Dmitriev. (6) Possibly place plasticine into the corners of the lid to raise the lid slightly above the sealed cover slips when closing the lid over the plates. (7) Store either at room temperature or at 4C (or at any other temperature that may be suggested by dynamic light scattering experiments; note that a defined, constant temperature can be very valuable for reproducibility). (8) Regularly check the setups and take detailed notes. More information can be obtained in the excellent textbooks by Bergfors [2] and Ducruix and Giegé [123]. 5.4. Good or bad precipitate? To tell apart a crystal from aggregated protein precipitate is an easy task. However, to distinguish between promising and bad precipitate is much more difficult. This is where experience makes all the difference. In order to get you on the right track for developing your own judgment skills, you might like to check out the excellent web site by Therese Bergfors at http:// xray.bmc.uu.se/~terese/crystallization/library.html. Once you are able to distinguish promising from bad conditions, you are on the right way to optimize the crystallization experiments. Good tips for optimization have been reviewed by D’Arcy [124] and Chayen [125] and, in a broader perspective, by Vincentelli et al. [9].
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Crystallography and Lectin Structure Database - CNRS

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