Cryo Trouble? Give the following tips a try next time your crystal is fussy about freezing.

Try X-ray data collection at room temperature.

Evaluate other cryoprotectants. Try CryoPro from Hampton Research, which contains 36 unique cryopreservation reagents.
For more information visit http://hamptonresearch.com/product_detail.aspx?cid=30&sid=189&pid=30

Mixing of different cryoprotectants can have a superior protective effect over single component cryoprotectants of the same total concentration.

冷冻麻烦？ 下次当您的水晶对冷冻很挑剔时，请尝试以下提示。

尝试在室温下收集 X 射线数据。

评估其他冷冻保护剂。 试试 Hampton Research 的 CryoPro，它包含 36 种独特的冷冻保存试剂。
欲了解更多信息，请访问 http://hamptonresearch.com/product_detail.aspx?cid=30&sid=189&pid=30

不同冷冻保护剂的混合比相同总浓度的单组分冷冻保护剂具有更好的保护效果。

Change the rate of cooling.  改变冷却速度。

a) Accelerate the rate of cooling. The fastest cooling rates have been achieved by blowing off the gas layer on liquid nitrogen during plunge cooling. (Hyperquenching for protein cryocrystallography, M. Warkentin et al, J. Appl. Cryst. (2006). 39, 805-811)

b) Slow the rate of cooling. Perhaps better suited to crystals with smaller solvent channels. The key to successful slow cooling of protein crystals is to carefully and completely remove all of the solvent from the surface of the crystal using oil such as Paratone-N, Perfluoropolyether, Mineral, Silicon, NVH or other. Remove ALL of the liquid from the surface of the crystals when using oil. (Slow cooling and temperature-controlled protein crystallography, Warkentin et al 10.1007/s10969-009-9074-y and Slow cooling of protein crystals, Warkentin et al, Volume 42, Part 5, Pages 944-952, October 2009) For more information visit http://hamptonresearch.com/menus.aspx?id=3&sid=138

a) 加快冷却速度。 通过在骤冷期间吹掉液氮上的气体层，可以实现最快的冷却速度。 （蛋白质低温晶体学的超淬灭，M. Warkentin 等人，J. Appl. Cryst. (2006). 39, 805-811）

b) 减慢冷却速度。 也许更适合溶剂通道较小的晶体。 成功缓慢冷却蛋白质晶体的关键是使用 Paratone-N、全氟聚醚、矿物、硅、NVH 或其他油等仔细彻底地去除晶体表面的所有溶剂。 使用油时，除去晶体表面的所有液体。 （缓慢冷却和温度控制的蛋白质晶体学，Warkentin 等人 10.1007/s10969-009-9074-y 和蛋白质晶体的缓慢冷却，Warkentin 等人，第 42 卷，第 5 部分，第 944-952 页，2009 年 10 月） 访问 http://hamptonresearch.com/menus.aspx?id=3&sid=138

Using salt as a crystallization reagent? Many salts are cryosalts, including malonate, formate, citrate, tartrate, acetate, Tacsimate and other organic acids, ammonium sulfate (>3.5 M), lithium sulfate, lithium chloride and other alkylammonium salts. For cryo try increasing your salt concentration by 20%. (Cryosalts: suppression of ice formation in macromolecular crystallography, K. A. Rubinson et al, Acta Cryst. (2000). D56, 996-1001 doi:10.1107/S0907444900007587.

Try the identical cryo procedure again with another crystal.

Vary the time and temperature of the crystal handling steps.

Check the liquid nitrogen level in your dewar and maintain a consistent level, day to day, week to week, month to month, year to year.

Try annealing. (Macromolecular crystal annealing: Techniques and cases studies. Bunick et al, The Rigaku Journal Vol. 15/ number 2/ 1998 and Macromolecular crystal annealing: overcoming increased mosaicity associated with cryocrystallography, Harp et al. (1998). Acta Cryst. D54, 622-8

使用盐作为结晶试剂？很多盐是冰晶盐，包括丙二酸盐、甲酸盐、柠檬酸盐、酒石酸盐、乙酸盐、Tacsimate和其他有机酸、硫酸铵（>3.5 M）、硫酸锂、氯化锂和其他烷基铵盐。对于冷冻，尝试将盐浓度提高 20%。 （冷冻盐：大分子晶体学中冰形成的抑制，K. A. Rubinson 等人，Acta Cryst. (2000). D56, 996-1001 doi:10.1107/S0907444900007587。

用另一个晶体再次尝试相同的冷冻程序。

改变晶体处理步骤的时间和温度。

检查杜瓦瓶中的液氮水平，并每天、每周、每月、每年保持一致的水平。

尝试退火。 （高分子晶体退火：技术和案例研究。Bunick 等人，理学杂志第 15 卷/第 2 期/1998 年和高分子晶体退火：克服与低温晶体学相关的镶嵌性增加，Harp 等人（1998 年）。Acta Cryst. D54， 622-8

Match the osmotic pressure of your cryoprotectant to the osmotic pressure of the reagent producing the crystal. Crystallization reagents with lower salt concentrations require a higher percentage of cryoprotectant for cryo protection than crystallization reagents with higher salt solutions (Cool data: quantity and quality. Elspeth Garman. Acta Cryst. (1999). D55, 1641-1653.). Osmolality tables (Weast, R. C. (1988-1989). Editor. Handbook of Chemistry; Physics, 69th ed. Boca Raton, Florida: CRC Press) can be used to estimate the osmolality of reagents. Another trick is to slowly concentrate a drop of the mother liquor by leaving the drop open to air and allowing the drop to slowly dry down, checking the mother liquor for clear glass freeze every few minutes. If you do not have X-rays to check for clear glass freeze, you can guesstimate by carefully placing the dewar under a dissecting microscope with overhead lighting. Focus on the surface of the liquid nitrogen, and bring the cooled loop into view just above the surface of the liquid nitrogen, where it is cold enough for guesstimate freezing.

使冷冻保护剂的渗透压与产生晶体的试剂的渗透压相匹配。与具有较高盐溶液的结晶试剂相比，具有较低盐浓度的结晶试剂需要更高百分比的冷冻保护剂来进行冷冻保护（酷数据：数量和质量。Elspeth Garman. Acta Cryst. (1999). D55, 1641-1653.）。渗透压表 (West, R. C. (1988-1989). Editor. Handbook of Chemistry; Physics, 69th ed. Boca Raton, Florida: CRC Press) 可用于估计试剂的渗透压。另一个技巧是慢慢浓缩一滴母液，方法是让一滴母液对空气敞开，让液滴慢慢干燥，每隔几分钟检查一次母液是否有透明玻璃结冰。如果您没有 X 射线来检查透明玻璃是否冻结，您可以通过将杜瓦瓶小心地放在带顶灯的解剖显微镜下进行猜测。专注于液氮的表面，将冷却回路置于液氮表面上方的视野中，那里足够冷，可以推测冻结。

Try high pressure cooling. (High-pressure cooling of protein crystals without cryoprotectants, Kim et al, Acta Cryst. (2005). D61, 881–890)

Cryoprotection of delicate crystals – Artem Evdokimov’s humble recipe.
http://www.xtals.org/crystal_cryo.pdf

Two words. Liquid propane.
http://cars9.uchicago.edu/biocars/pages/flashcooling.shtml

Roger S. Rowlett’s Cryoprotectant Strategy
1. Transfer crystals to mother liquor plus 30%v/v glycerol or ethylene glycol (sometimes lower depending on crystallization
reagent).
2. Transfer crystals to mother liquor plus 30%w/v glucose (or try sequential soaks in mother liquor plus 15%w/v and then 30%w/v glucose. Just a few seconds or minutes is usually enough). Glucose or other sugars often work when glycerol or ethylene glycol fails.
3. Try the “no-fail” in situ cryo method, which is a gradual buildup of cryoprotectant. 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.
a) Prepare a solution of artificial mother liquor plus 30% w/v glucose (40% v/v glycerol or another cryoprotectant can be substituted)
b) 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.
c) 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.
d) 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.
This is very gentle, and often works when #1 and #2 does not, but in our hands nearly always increases mosaicity. (But mosaic is better than no diffraction.)
4. Try dragging crystals though Paratone-N to remove surface water from the crystal. This actually nearly always works for us, but is more fussy than #1 or #2, and it is easier to damage crystals during manipulation because of the viscosity of the oil.
I normally plunge protected crystals into liquid nitrogen after mounting.
Ice rings are a good indication of poor cryoprotection, but lack of diffraction could just be your crystals, too. For our latest dataset, we just sorted through 38 crystals until we found a good one. The key, as it turned out, is that all of our beautiful large crystals were apparently difficult to visualize disordered stacks of plates (we didn’t notice this until some fractured during cryo-soaks) whereas some of the small crystals were actually single crystals. We selected a decently diffracting small one and took loooooooooooong frames to get a good data set. (Roger S. Rowlett, Professor, Colgate University Presidential Scholar, Department of Chemistry, Colgate University, NY, USA)

Dig around in the cryoprotectant database for ideas. http://idb.exst.jaxa.jp/db_data/protein/search-e.php

Try 10% glycerol. (Jim Pflugrath)

Try 10 to 20% 2,3-butanediol. It can also reduce mosaicity. Try a quick dip, 30 to 60 seconds.

Fine tune the cryo concentration by screening 2.5% concentration increments, dipping a loop in the cryo buffer, freezing it and collecting X-Ray diffraction data to find the minimum concentration required that produces a clear glass and no ice rings.

Try dragging the crystal through a 1:1 mix of Paratone-N and Mineral oil until most or all the mother liquor from surrounding the crystal has been removed. (David Briggs)

Look back in your screen plates for a different crystal in different reagent, do not pass go, do not collect $200.00 and start over at the top of this tip.

Roger S. Rowlett 的冷冻保护剂策略
1. 将晶体转移到母液中，加入 30%v/v 甘油或乙二醇（有时根据结晶情况降低
试剂）。
2. 将晶体转移到加 30%w/v 葡萄糖的母液中（或尝试在母液中依次浸泡加 15%w/v 和 30%w/v 葡萄糖。通常只需几秒钟或几分钟就足够了）。当甘油或乙二醇失效时，葡萄糖或其他糖通常会起作用。
3. 尝试“不失败”的原位冷冻方法，这是一种逐渐积累的冷冻保护剂。这种方法特别适用于不能直接转移到冷冻保护剂溶液中的晶体，或对液滴蒸发驱动的母液浓度变化特别敏感的晶体。在我们的实验室中，这种方法通常成功地用于其他非常敏感的晶体。这种特殊的方法适用于悬滴结晶。如果在冷冻保护剂溶液中以最终所需浓度的 125% 包含配体，则可以在冷冻保存的同时浸泡配体。
a) 制备人工母液加 30% w/v 葡萄糖的溶液（可以用 40% v/v 甘油或其他冷冻保护剂代替）
b) 取出含有一滴需要冷冻保护的晶体的盖玻片，加入 0.25 滴体积 (DV) 的冷冻保护剂溶液（例如，对于 4 uL 的液滴，加入 1 uL 的冷冻保护剂溶液）。盖好盖玻片，静置 5 分钟。检查晶体是否破裂和/或溶解。
c) 重复上一步，添加以下额外的冷冻保护剂：0.25 DV、0.50 DV、1.00 DV、2.00 DV。每次添加后，更换孔上的盖玻片并静置 5 分钟。检查晶体是否开裂和/或溶解。
d) 最后一次添加和孵育 5 分钟后，取下盖玻片，用固定环捞出晶体并直接在液氮中冷冻。最终的葡萄糖浓度为 24%，足以保护大多数结晶溶液在液氮中冷冻时不会结冰。
这是非常温和的，并且通常在 #1 和 #2 不起作用时起作用，但在我们手中几乎总是会增加镶嵌性。 （但马赛克总比没有衍射要好。）
4. 尝试通过 Paratone-N 拖动晶体以去除晶体中的表面水。这实际上几乎总是对我们有用，但比 #1 或 #2 更挑剔，并且由于油的粘度，在操作过程中更容易损坏晶体。
我通常在安装后将受保护的晶体放入液氮中。
冰环是低温保护不良的一个很好的迹象，但缺乏衍射也可能只是你的晶体。对于我们最新的数据集，我们只是对 38 个晶体进行分类，直到找到一个好的晶体。事实证明，关键是我们所有美丽的大晶体显然很难看到无序的叠层板（直到在低温浸泡过程中有些破裂我们才注意到这一点），而一些小晶体实际上是单晶.我们选择了一个像样的衍射小帧，并用了 looooooooooooong 帧来获得一个好的数据集。 （Roger S. Rowlett，美国纽约州科尔盖特大学化学系科尔盖特大学校长学者教授）

在冷冻保护剂数据库中四处寻找想法。 http://idb.exst.jaxa.jp/db_data/protein/search-e.php

试试 10% 的甘油。 （吉姆·普弗拉格拉斯）

尝试使用 10 至 20% 的 2,3-丁二醇。它还可以减少镶嵌性。尝试快速浸泡 30 到 60 秒。

通过筛选 2.5% 的浓度增量来微调低温浓度，在低温缓冲液中浸一个循环，将其冷冻并收集 X 射线衍射数据，以找到产生透明玻璃和无冰环所需的最低浓度。

尝试将晶体拖过 Paratone-N 和矿物油的 1:1 混合物，直到去除晶体周围的大部分或全部母液。 （大卫布里格斯）

在您的筛板中查看不同试剂中的不同晶体，不要错过，不要收取 200.00 美元，然后从本提示的顶部重新开始。