Using Additives with Oryx6/8

We are often asked for suggestions for the use of additives with Oryx6/8 to improve the morphology or order of crystals. Once crystallization conditions have been found, additives can perhaps most easily be tested as follows:

1. Design an XSTEP spreadsheet where every well is identical and contains the appropriate crystallization conditions (allowing for the addition of 0.2µl of additive which follows)
2. Execute this spreadsheet into the first 24 wells of a Nunc HLA plate
3. Dispense 100 µl of each of 24 additives in the wells of Nunc Strips (as is done with screening solutions).
4. Use the ASPRUN software package to transfer 0.2µl of each additive into the wells that were previously dispensed.

Note that 0.2µl is about the smallest quantity that can be transferred reliably. If problems arise through excessive nucleation or precipitation, dispense at a lower concentration and use the D’Arcy evaporation technique to increase the concentration.

Here are some suggestions for additives, followed by comments on their sources and effects. The concentrations of the stock solutions are shown (i.e. before dilution by a factor of 10).

1. b-octyl glucoside 3%
2. Heptane-1,2,3 triol 4%
3. Spermine 0.1M
4. Hexanediamine 0.1M
5. Malonate pH7.0 0.1M
6. Glycine 1M
7. Taurine 0.1M
8. Betaine 0.1M
9. Trimethylamine n-oxide 0.1M
10. polyvinylpolypyrrolidone 5%
11. 2-Methyl 2,4-pentanediol 20%
12. Co hexamine chloride 0.5%
13. CsCl 0.1M
14. Hexanediol 0.1M
15. Xylitol 50%
16. Sucrose 50%
17. Phenol 0.1M
18. Thymol 0.1M
19. Urea 0.1M
20. KSCN 0.1M
21. Dioxane 20 %
22. CaCl 0.1M
23. Mg acetate 0.1M
24. Mn acetate 0.05M
25. Co acetate 0.05M
26. Zn acetate 0.05M
27. Cd acetate 0.05M
28. Ba acetate 0.05M
29. Pr acetate 0.05M
30. Sm or Eu acetate 0.05M
31. Yb acetate 0.05M
32. Ca acetate 0.1M

Additives 1-20 were suggested by Dr. E. Conti (Imperial College, London) partly based on reagents examined by C.H.Schein (Biotechnology, 8, 308-317 (1990)). Dr. J. Fonticilla-Camps (IBS, Grenoble) suggested additive 21, dioxane, for cases where there is aggregation of the protein. Dr. J. Jankaric (U.C. Berkeley, CA) suggested additive 22, CaCl, to improve the order of poorly-diffracting crystals.

Yaakov Korkin at the Weismann Institute, Israel, suggested additives 23-32 to improve poorly-diffracting crystals. These are based on ideas from Paul Sigler’s Laboratory at Yale University. The acetates are suggested because they are generally very soluble, but if an anion is already present at high concentrations it should preferably be used instead eg. MgCl₂, MgSO₄ etc.). This will reduce the number of variables. In addition, Korkin suggested Ca²⁺ to increase nucleation.

Please send suggestions for other additives and their effects to Douglas Instruments.

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