Analysis Of 2d Crystallization Reactions

Even if 2D crystals of large membrane proteins can sometimes be observed by light microscopy, the shape of the crystals and their degree of order can best be assessed by electron microscopy. Thus, screening of 2D crystallization experiments requires access to an electron microscope. The quickest specimen preparation method for screening for 2D crystals is negative staining. This method is rapid, needs a small amount of sample (less than 5 pL), is remarkably simple, and can provide structural information to a resolution of about 2 nm. A negatively stained 2D crystal is embedded in a dry microcrystalline heavy atom replica. As heavy atoms used for negative staining scatter electrons much more than biological atoms (C, H, O, N, P, and S), the contrast is drastically increased but also inverted (hence the term "negative" staining). In addition, the heavy atom salts partially substitute the water in the native environment of the molecules, thus embedding the specimen and protecting it from collapse upon drying in air. The most commonly used heavy atom salts are uranyl acetate or formate and sodium or potassium phosphotungstate. Uranyl salts are more suitable for more protein samples, whereas tungstate is useful for lipid structures. The pH of tungstate solutions can be modified, whereas uranyl salts precipitate at pH greater than 4.5.

After negative staining, examination of the samples is carried out by transmission electron microscopy. The presence of large vesicles or sheets can be checked at low magnification (2000x-5000x) because of the high contrast obtained when operating at large underfocus even for very thin objects (Figure 5A). Further observations are done at higher magnification (Figure

Figure 5. Negatively stained tubular crystals of PSII complex obtained as described in Reference

47. (A) Low magnification (5000x). Scale bar represents 500 nm. (B) High magnification (50 000x). Scale bar represents 100 nm.

Figure 5. Negatively stained tubular crystals of PSII complex obtained as described in Reference

47. (A) Low magnification (5000x). Scale bar represents 500 nm. (B) High magnification (50 000x). Scale bar represents 100 nm.

5B). If no crystals are found, the presence of remaining detergent, single protein particles incorporated in lipid vesicles, solubi-lized proteins, or protein aggregates can all be identified. This information may be valuable for designing further crystallization trials.

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