Cryo

Introduction

Cryopreservation of protein crystals has at least two advantages over room temperature methods. First, it greatly reduces radiation damage of the crystallized protein, especially when irradiated with higher intensity radiation sources. Second, it provides for relatively simple storage and transportation of crystals for remote data collection. Typical cryopreservants include glycerol, sugars (glucose and sucrose), and polyethylene glycols. Cryopreserved crystals are usually stored at liquid nitrogen temperatures (77K). Cryopreservants prevent the formation of ice in the cooled crystal and supporting mother liquor; instead of freezing when cooled, cryopreserved solutions vitrify into clear, supercooled glasses. Glycerol (30%) or glucose (25%) is usually sufficient to cryoprotect most crystallized proteins. Lower concentrations of cryoprotectants are necessary in the presence of high concentrations of salts or polyethylene glycol. Generally, if a drop of well solutions vitrifies to a clear glass in a sample loop, the cryoprotectant concentration is sufficient for ice formation suppression. The simplest method of cryoprotection is to simply transfer crystals directly from their mother liquor to a drop of artificial mother liquor with the added cryoprotectant. Soaking in the cryoprotectant drop for as little as 30 seconds is usually sufficient to prevent ice formation or crystal cracking. Longer soaks may be required if protein ligands are to be soaked in at the same time.

No-fault Cryoprotection

This method is especially appropriate for crystals that cannot tolerate direct transfer to cryoprotectant solution, or for crystals that are especially sensitive to concentration changes in the mother liquor driven by drop evaporation. In our laboratory this method is routinely used with success on otherwise very sensitive crystals. This particular method is adapted for hanging drop crystallization. Ligands can be soaked in at the same time as cryopreservation if included in the cryoprotectant solution at 125% of the final, desired concentration.

• Prepare a solution of artificial mother liquor + 30% w/v glucose (40% v/v glycerol or another cryoprotectant can be substituted)
• Remove a coverslip containing a drop with crystals to be cryoprotected and add 0.25 drop volume (DV) of cryoprotectant solutions (e.g. for a 4 uL drop add 1 uL of cryoprotectant solution). Replace coverslip on well and let stand for 5 minutes. Examine the crystals for cracking and/or dissolution.
• Repeat the previous step with the following additional cryoprotectant additions: 0.25 DV, 0.50 DV, 1.00 DV, 2.00 DV. After each addition replace the coverslip over the well and let stand for 5 minutes. Examine crystals for cracking and/or dissolution.
• After the last addition and 5 minute incubation, remove coverlip, fish out crystals with mounting loops and freeze directly in liquid nitrogen. The final glucose concentration will be 24%, sufficient to protect most crystallization solutions from ice formation upon freezing in liquid nitrogen.

protocol contributed by Roger Rowlett, Colgate University Department of Chemistry

Freezing in liquid Propane

It is proposed that freezing in liquid propane is faster and therefore better for a protein than in liquid nitrogen. Measurements with small thermocouples by Hakan Hope [1]revealed that there is almost no difference in cooling rates; cooling in liquid nitrogen was even faster than in liquid propane. On the other hand Teng and Moffat [2] showed that flash cooling in liquid propane is fastest. In summary, some but not all crystals freeze 'better' in liquid propane than in nitrogen. Transport of crystals embedded in solid propane is easier than in liquid nitrogen: you can take your dry-shipper with you without any liquid nitrogen left in the container. Here a short protocol how to prepare liquid propane and how to use it.

• Cryogenic burns are painful. E.g. wearing a combination of cotton gloves and latex gloves protects from liquid propane and are not too bulky for crystal handling.
• Use pure propane
• Place a metal piece (e.g. big screw nut) into a 50 ml plastic ('Falcon') tube
• Put the plastic tube with metal piece into liquid nitrogen. Fix it with clamps or similar. Take care that no liquid nitrogen is in the tube.
• Connect a tubing with a Pasteur pipette at the end to the valve on the propane cylinder.
• Put Pasteur pipette into the plastic tube with the metal piece. Propane will condensate on the cool metal piece.
• After 0.5-1 min you should have 20-30 ml of propane.
• If you want to store the propane leave it in the liquid nitrogen until it is solid. This might take a few minutes.
• Fill the liquid propane into vials. For short time storage and crystal handling put the vials in a flat liquid nitrogen bath. If propane becomes solid after a while just remove the vial from the liquid nitrogen and put it on the desk; let it stand for a while until it is liquid again and put it pack into the nitrogen bath.
• Plunge the crystals just into the vials and wait until the propane is solid.

Salts as precipitants ...

Some salts may also serve as cryoprotectants: malonate (see Acta Cryst D59, 2356), formate, ammonium sulfate (at >3.5 M), lithium sulfate, and perhaps others. Anything with a flat solubility vs temperature is a good candidate. Mixing of different cryos can often have a superior protective effect to single-component cryos of the same total concentration (the "confusion principle"), so there are a lot of combinations to try.

See also

• Cryoprotectant database used to be at http://idb.exst.jaxa.jp/db_data/protein/search-e.php? or http://idb.exst.jaxa.jp/db_data/protein/200304E02478000.html ; still to be found at http://web.archive.org/web/20111011202903/http://idb.exst.jaxa.jp/db_data/protein/search-e.php
• see concentrations given in: McFerrin and Snell, J.Appl.Cryst (2002) 35, 538 and Mitchell and Garman, J.Appl.Cryst. (1996) 29, 584
• faster freezing: a simple means (blowing away the gas layer) is described by Warkentin et al. (2006). J. Appl. Crystallogr. 39, 805.
• annealing: e.g. Harp, J., Timm, D. & Bunick, G. (1998) Macromolecular crystal annealing: overcoming increased mosaicity associated with cryocrystallography. Acta Cryst. D54, 622-8; Yeh, J. & Hol, W. (1998) A flash-annealing technique to improve diffraction limits and lower mosaicity in crystals of glycerol kinase. Acta Cryst. D54, 479-80; Kriminski, S., Caylor, C.; Nonato, M., Finkelstein, K. & Thorne, R. (2002) Flash-cooling and annealing of protein crystals. Acta Cryst. D58, 459-71
• slow cooling: Warkentin, M. & Thorne, R. E. (2009) Slow cooling of protein crystals. J Appl Cryst. 42, 944-952
• This cryocrystallography webinar lists some common cryoprotectants: http://www.rigaku.com/protein/webinar-001.html