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Cryo: Difference between revisions
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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 [http://www.bio.net/bionet/mm/xtal-log/1995-November/002005.html]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 [http://scripts.iucr.org/cgi-bin/paper?wb0048] 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. | 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 [http://www.bio.net/bionet/mm/xtal-log/1995-November/002005.html]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 [http://scripts.iucr.org/cgi-bin/paper?wb0048] 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. | 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 | * 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 | * Use pure propane | ||
* Place a metal piece (e.g. big screw nut) into a 50 ml plastic ('Falcon') tube | * Place a metal piece (e.g. big screw nut) into a 50 ml plastic ('Falcon') tube | ||
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* 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. | * 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. | * 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 | |||