Have a crystal grown in the Hampton Research SaltRx Screen reagent with less than 1 M salt and are wondering what cryo condition to try?

We would recommend starting with 30% v/v Glycerol in the drop as a cryoprotectant and adjust the concentration as needed based on diffraction data.

The CryoPro user guide is a nice overview of how to select a cryogen if you’re willing to try something beyond glycerol. 

In the Crystallization Tips section of the Hampton Research web site there are some tips for cryogens.  Here are a few.  Hope this helps.

Cryo buffer

We've had good success using the well solution directly as the foundation of a cryobuffer in several situations where crystals cannot be grown directly in the presence of cryoprotectant, and where crystals don't tolerate transfer to artificial mother liquors. The basic protocol is as follows:

1. Remove 100 microliters of the well solution after crystals have grown

2. Split this sample into two 50 microliter aliquots.

3. Add 7.5 mg of dextrose (glucose) to the first aliquot and 15 mg of dextrose to the second. Dissolve by gentle pipeting with a wide-bore tip. This will give two sequential well solutions that now contain 15% and 30% w/v dextrose. If all the dextrose won't go into the second aliquot, spin hard and remove the supernatant.

4. Transfer the crystal to aliquot 1, equilibrate for 3 minutes, then to aliquot number 2, then freeze. We've had a few crystals that routinely crack or blow up when transfered to artificial mother liquor that behave well when transfered to well solution plus glucose. We assume that there is some aspect of the crystal drop (pH, ionic tension, precipitant concentration) that is more effectively reproduced within the well than by separately prepared mother liquors. The nice thing about the protocol above is that you don't get much of a volume increase when dry dextrose is dissolved in the well solution, so the components in the solution are not diluted.

Finally, if you don't get a really good freeze, you can try to add about 5% v/v glycerol to aliquot 2 in addition to the 30% w/v dextrose. Reference: Personal communication from Barry Stoddard, Fred Hutchinson Cancer Research Center

Cryo for Ammonium sulfate and salt based crystallization reagents

Increase the concentration of ammonium sulfate (cryosalt) (or primary salt acting as a precipitant) in increments of 10% in the presence of 5 to 10% v/v Glycerol.

Evaluate 25-30% w/v glucose, or trehalose or sucrose as cryoprotectants for crystals grown in Ammonium sulfate. Acta Cryst. (2002). D58, 1664-1669. Crystallization of RNA/protein complexes. M. Garber, G. Gongadze, V. Meshcheryakov, O. Nikonov, A. Nikulin, A. Perederina, W. Piendl, A. Serganov and S. Tishchenko.

15 to 30% v/v Ethylene glycol, DMSO, or Glycerol.

Try 1 – 2 M Sodium malonate as a cryoprotectant. Acta Cryst. (2003). D59, 2356-2358. Malonate: a versatile cryoprotectant and stabilizing solution for salt-grown macromolecular crystals. T. Holyoak, T. D. Fenn, M. A. Wilson, A. G. Moulin, D. Ringe and G. A. Petsko.

Place a drop of 75% v/v Paratone-N, 25% v/v Paraffin Oil next to the drop containing the crystal. Remove the crystal from the drop using a CryoLoop and dip the crystal into the oil. Keeping the CryoLoop and crystal immersed, gently move the CryoLoop containing the crystal from the oil into the reagent, back into the oil, back into the reagent, repeating this several times to remove some of the aqueous (reagent) layer from the crystal surface. Cryo cool the crystal.

Practical macromolecular cryocrystallography

Practical macromolecular cryocrystallography by J.W. Pflugrath

Acta Crystallogr F Struct Biol Commun. 2015 Jun 1; 71(Pt 6): 622–642. Published online 2015 May 27.

Abstract

Cryocrystallography is an indispensable technique that is routinely used for single-crystal X-ray diffraction data collection at temperatures near 100 K, where radiation damage is mitigated. Modern procedures and tools to cryoprotect and rapidly cool macromolecular crystals with a significant solvent fraction to below the glass-transition phase of water are reviewed. Reagents and methods to help prevent the stresses that damage crystals when flash-cooling are described. A method of using isopentane to assess whether cryogenic temperatures have been preserved when dismounting screened crystals is also presented.