Divalent Metals

Divalent metals are used for the crystallization of various enzymes. The role of these metals in crystallization can often be questioned.
Metals required for function
For protein which require such metals for activity, their use is quite clear. It is important to investigate substitutions by one metal for another. Such changes may result in important changes that mat lead to improved crystals. The concentrations to be used will probably be closer to the lower limit given in the tables.
DNA and RNA crystallization
Magnesium, calcium, cobalt hexamine are commonly used in the crystallization of nucleic acids and should be investigated with respect to their concentration. The amount of magnesium added during annealing should be kept to a minimum, so that the range of divalent metals and their concentration which can be changed later during the crystallization can be maximized.
Buffers and Precipitants The phosphates and some sulfates of divalent metals have poor solubility. So, avoid the use of such salts. Ammonium sulfate, phosphate buffer and divalent metals is the worse possible combinations. Try for yourself in the absence of any protein, so that you may identfy the crystals when they occur in your protein drop. Citrate, malate and other compounds in this series have similar properties. Succinate is a little better, but it is better to chose a buffer that will not chelate the metals. Cacodylate, acetate are good candidates. Chelators
Imidazole, EDTA, EGTA are good chelators. Their use in small concentrations as a secondary additive, may allow greater control over the effect of the divalent metals as it might help against non specific binding, while being unable to compete with specific binding. Their effect should be analysed properly. To ensure that this is done carefully, working solutions should be used. The chelator should be analysed as a perturbation to the divalent metal containing working solution, so that the effect of the chelator and the optimum concentration can be analysed, independently of random variations in divalent metal concentrations that may occur as a result of mixing errors.

Specific Metals

Metals like zinc, (copper and cobalt) have a tendency to bind to non-specific sites. They should never be used in large quantities. They are also more likely to give salt crystals. Hence they are excellent to determine if the unknown buffer your protein comes in contains phosphate. Please note the word PBS (PBS = phosphate buffered saline, typically 100-250 mM NaCl, 5-50 mM Phosphate, pH 7-7.5) that may be somewhere on the label. The zinc concentration in Hampton screen I (condition 45: 0.2M zinc acetate, 18% PEG 8,000, 0.1M Na cacodylate, pH 6.5) is at the limit of what is recommended. It is quite prone to give salt crystals and aggregate susceptible proteins. Instead of wasting protein check condition 27 from Hampton screen II first (0.01 zinc sulfate, 25% MPEG 550, 0.1 MES, same pH, 6.5).
Check the pH of the solution when you use high zinc concentration, you may find that it has changed, and what you are observing is a consequence of pH change and not the effect of zinc.
Copper is even more problematic than zinc. All that is said for zinc is more true for copper. In addition it can in some cases cleave proteins "Specific cleavage of immunoglobulin G by copper ions." Smith et al., Int J Pept Protein Res (1996) 48:48-55; modify DNA and cleave RNA.

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Updated/Created on Sep-23-97 by EAS, TSRI