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Additional info for Macromolecular Cyrstallography Protocols Vol.2: Structure Determination (Methods in Molecular Biology Vol 364)
When using prefrozen crystals, we have had good results with cryotongs that have been modiﬁed for the tight helium cryostream geometry. If, when mounting, nitrogen from LN2 freezes on the sample, it can be gently removed by direct manipulation with an unused cryoloop. 4. Heating from the photolysis and illumination beams can be signiﬁcant. For photolysis, low-intensity illumination protocols should be used; heating in excess of 10–20°C occurs with direct illumination with a laser. If monochromatic illumination must be used, a standard trick is to choose a long wavelength where the crystal appears optically less thick.
1. and mount crystals directly in the loop of a CryoCap. 2. Freeze the crystals directly in the helium stream. Note that prefreezing in liquid nitrogen is not possible. 3. Illuminate the crystal with low-intensity light, such as a ﬁber-optic illuminator (see Note 4). Collection of data should be performed with the illumination light on. 2K, so samples should be under continuous illumination at low power during data collection. Simultaneously, too high an illumination will result in signiﬁcant crystal heating and increased recombination (17).
0. 16. 0. 17. 0. 18. XI “adequate” cryoprotection buffer: 100 mM Tris-HCl, 2 mM MgCl2, 30% (w/v) (NH4)2SO4, serial passage through solutions of this material with 5–30% glycerol (w/v). 19. 5 M xylose, and 40% (w/v) (NH4)2SO4. 20. 5, 8–10% polyethylene glycol 3350. 21. 5, 8–10% polyethylene glycol 3350, serial passage through solutions of this material with 5 and 20% glycerol (w/v). 3. Methods Detailed next are three methods for annealing crystals that resulted in two successes and one failure. The unsuccessful outcome is included to assist in determining the conditions when any annealing protocol will not result in improved diffraction characteristics (see Note 4).