分类目录归档：Hampton蛋白结晶技术与建议

Cryo buffer 冷冻缓冲液

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Cryo buffer 冷冻缓冲液

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

冷冻缓冲液
在几种情况下，在存在冷冻保护剂的情况下晶体不能直接生长以及晶体不能转移到人工母液中的情况下，我们直接使用井溶液作为冷冻缓冲液的基础取得了很好的成功。基本协议如下：

1. 晶体生长后取出 100 微升孔溶液

2. 将此样品分成两份 50 微升的等份。

3. 在第一个等分试样中加入 7.5 mg 葡萄糖（葡萄糖），在第二个等分试样中加入 15 mg 葡萄糖。用大口径吸头轻轻吹打溶解。这将给出两个顺序井解决方案，现在包含 15% 和 30% w/v 葡萄糖。如果所有的葡萄糖都不会进入第二个等分试样，请用力旋转并去除上清液。

4. 将晶体转移到 1 号等分试样中，平衡 3 分钟，然后转移到 2 号等分试样中，然后冷冻。我们有一些晶体在转移到人造母液中时会经常破裂或爆炸，当转移到井溶液和葡萄糖中时表现良好。我们假设晶体液滴的某些方面（pH、离子张力、沉淀剂浓度）在井内比单独制备的母液更有效地再现。上述协议的好处是，当干葡萄糖溶解在孔溶液中时，体积不会增加太多，因此溶液中的成分不会被稀释。

最后，如果您没有得到很好的冷冻效果，除了 30% w/v 葡萄糖外，您可以尝试在等分试样 2 中添加约 5% v/v 甘油。参考：来自 Fred Hutchinson 癌症研究中心的 Barry Stoddard 的个人交流

A is for Aggregation 阻止包括蛋白质、肽和核酸在内的生物大分子的结晶

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A is for Aggregation  A 代表聚合

Aggregation can be a deterrent to the crystallization of biological macromolecules including proteins, peptides, and nucleic acids. The presence of sample aggregation can be detected by either dynamic light scattering or native gel electrophoresis. Aggregation might be caused by hydrophobic patches on the surface of the sample, differently charged isoforms, differently phosphorylated isoforms, mixtures of methylated and non-methylated samples, glycosylation, as well as electrostatic interactions. Aggregation can be due to autologous aggregation where the protein is aggregating with itself or heterologous contamination where the sample is aggregating with other proteins. In the case of heterologous contamination, further purification of the sample should be seriously considered. In the case of autologous aggregation that precludes crystallization one might consider:

Using molecular biology to manipulate intra and inter molecule interactions by modifying the sample sequence (alter, add, or delete residues).

Use chemical additives to manipulate sample-sample and sample solvent interactions.
Detergents
Chaotropes (urea, guanidine hydrochloride, hydrochloric acid, etc)
Electrostatic agents
Alcohols (isopropanol, methanol, ethanol, etc)
Salts (sodium chloride, potassium chloride, sodium fluoride, etc)
Polyols (glycerol, PEG 400, etc)
Ligands, inhibitors, co-factors, and metals
Use temperature to prevent aggregation (0°C and 60°C)
Consider a fusion protein
Remove C-terminus or N-terminus
Truncate domains
Remove His-tag

In some cases aggregates can be removed by centrifugation or filtration.

In some cases the aggregates can be removed by mixing the sample with the crystallization reagent, allowing the sample to incubate for 15 minutes, centrifuging the sample/reagent mixture, removing the precipitate and setting the drop with the supernatant.

聚集可以阻止包括蛋白质、肽和核酸在内的生物大分子的结晶。可以通过动态光散射或天然凝胶电泳检测样品聚集的存在。聚集可能是由样品表面的疏水斑块、不同电荷的异构体、不同的磷酸化异构体、甲基化和非甲基化样本的混合物、糖基化以及静电相互作用引起的。聚集可能是由于蛋白质与自身聚集的自体聚集或样品与其他蛋白质聚集的异源污染。在异源污染的情况下，应认真考虑样品的进一步纯化。在排除结晶的自体聚集的情况下，可以考虑：

使用分子生物学通过修改样品序列（改变、添加或删除残基）来操纵分子内和分子间的相互作用。

使用化学添加剂来控制样品-样品和样品溶剂的相互作用。
洗涤剂
离液剂（尿素、盐酸胍、盐酸等）
静电剂
醇类（异丙醇、甲醇、乙醇等）
盐类（氯化钠、氯化钾、氟化钠等）
多元醇（甘油、PEG 400 等）
配体、抑制剂、辅因子和金属
使用温度防止聚集（0°C 和 60°C）
考虑融合蛋白
移除 C 端或 N 端
截断域
删除 His 标签

在某些情况下，可以通过离心或过滤去除聚集体。

在某些情况下，可以通过将样品与结晶试剂混合、让样品孵育 15 分钟、离心样品/试剂混合物、去除沉淀物并用上清液放置液滴来去除聚集体。

Trouble with protein storage 蛋白质储存问题

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Trouble with protein storage 蛋白质储存问题

Try 3 to 5% 1,2-propanediol in the protein buffer as a substitute for glycerol to stabilize proteins. Annie Hassell, Glaxo Smithkline 2009

尝试在蛋白质缓冲液中加入 3% 到 5% 的 1,2-丙二醇作为甘油的替代品来稳定蛋白质。 安妮哈塞尔，葛兰素史克 2009

Dissolving hydrophobic additives into oil-用于静滴蒸汽扩散或油下微量

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Dissolving hydrophobic additives into oil-用于静滴蒸汽扩散或油下微量

Try dissolving the small molecule additive into paraffin or silicon oil, and use this mixture to cover the sample drop. This can be used with sitting drop vapor diffusion or with microbatch under oil. The oil acts as a reservoir that may contain excess small molecule that (you hope) will be fed into the crystals.

将疏水性添加剂溶解到油中
尝试将小分子添加剂溶解在石蜡或硅油中，然后用这种混合物覆盖样品液滴。 这可用于静滴蒸汽扩散或油下微量。 油充当储存器，可能含有过量的小分子（您希望）将被送入晶体中。

硫代硫酸钠防止分子间二硫键 -促进晶体生长和避免形成不稳定和弱衍射晶体

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Sodium thiosulfate to prevent intermolecular disulfide bridges

The presence of thiosulfate in the protein solution was essential to promote crystal growth and to avoid the formation of unstable and weakly diffracting crystals(1); this is likely to be a consequence of the intrinsic capability of the reduced thiol group of the active-site cysteine to form disulfide bridges, leading to the destabilization of the protein native structure. Sulfane sulfur-donor compounds such as Na2S2O3 are likely to either keep the protein in the persulfurated form or to prevent intermolecular disulfide bridges leading to unfolding and aggregation

硫代硫酸钠防止分子间二硫键
蛋白质溶液中硫代硫酸盐的存在对于促进晶体生长和避免形成不稳定和弱衍射晶体至关重要 (1)；这可能是活性位点半胱氨酸的还原硫醇基团形成二硫键的内在能力的结果，导致蛋白质天然结构的不稳定。硫烷硫供体化合物（如 Na2S2O3）可能使蛋白质保持过硫化形式或防止分子间二硫键导致展开和聚集 。

(2). References 参考文献

1. Crystallization and preliminary crystallographic characterization of LmACR2, an arsenate/antimonate reductase from Leishmania major. D. Bisacchi, Y. Zhou, B. P. Rosen, R. Mukhopadhyay and D. Bordo. Acta Cryst. (2006). F62, 976-979. 2. Bordo, D., Forlani, F., Spallarossa, A., Colnaghi, R., Carpen, A., Bolognesi, M. & Pagani, S. (2001). Biol. Chem. 382, 1245–1252.

Benzylkonium chloride-膜蛋白和可溶性蛋白用阳离子表面活性剂

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Benzylkonium chloride-膜蛋白和可溶性蛋白用阳离子表面活性剂

Try benzylkonium chloride (Fluka 12060). This cationic surface active agent has been reported to be useful as a crystallization additive with membrane proteins and may be useful for soluble proteins. We’ve been using it in the drop between 1 and 3% w/v in water. Try a 10% w/v stock solution in water and dilute into the drop to 1-3%.

苄基氯铵
试试苄基氯铵 (Fluka 12060)。 据报道，这种阳离子表面活性剂可用作膜蛋白的结晶添加剂，并可用于可溶性蛋白。 我们一直在水中使用 1% 到 3% w/v 的浓度。 尝试在水中使用 10% w/v 的储备溶液，然后将其稀释至 1-3%。

Crystal Screen as an additive screen 附加屏幕

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Crystal Screen as an additive screen 附加屏幕

Although Hampton Research does offer a specifically formulated Additive Screen (HR2-428), here is a tip when one already has a sparse matrix screen or two laying about the lab. When screening additives try adding 50 microliters of each Crystal Screen reagent to 950 microliters the “best” crystallization conditions thus far in order to see if any of the reagents in Crystal Screen might serve as good additives. Crystal Screen 2 is an especially good kit for this technique since Crystal Screen 2 contains numerous divalent cations, Jeffamine® Reagent, and a few other “novel” agents. Jeffamine® is a registered trademark of the Huntsman Petrochemical Corporation

Reference 参考文献

Crystallization of foot-and-mouth disease virus 3C protease: surface mutagenesis and a novel crystal-optimization strategy. Acta Crystallogr D Biol Crystallogr. 2005 May;61(Pt 5):646-50. Epub 2005 Apr 20. Birtley JR1, Curry S.

水晶屏幕作为附加屏幕
尽管 Hampton Research 确实提供了一种专门配制的添加剂屏幕 (HR2-428)，但当一个人在实验室周围已经有一个或两个稀疏矩阵屏幕时，这里有一个提示。 当筛选添加剂时，尝试将 50 微升的每种 Crystal Screen 试剂添加到迄今为止“最佳”结晶条件的 950 微升中，以查看 Crystal Screen 中的任何试剂是否可以作为良好的添加剂。 Crystal Screen 2 是该技术特别好的套件，因为 Crystal Screen 2 包含大量二价阳离子、Jeffamine® 试剂和一些其他“新型”试剂。 Jeffamine® 是 Huntsman Petrochemical Corporation 的注册商标。

 

Glycerol  蛋白结晶蒸汽扩散结晶实验中添加甘油

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Glycerol  蛋白结晶蒸汽扩散结晶实验中添加甘油

Looking for yet another additive to try during optimization? Try glycerol. Typical concentrations in the drop can range from 3 to 15%. Glycerol may limit non-specific aggregation and could be useful if you plan to use cryo techniques during data collection.

When adding glycerol to the vapor diffusion crystallization experiment be sure to have a higher glycerol concentration in the reservoir than the drop otherwise you may find that your drops will get biggger and not smaller. Reference: Vera,L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011) Practical Use of Glycerol in Protein Crystallization. Cryst. Growth & Des. 11 :2755–2762

甘油
正在寻找另一种在优化过程中尝试的添加剂？ 试试甘油。 液滴中的典型浓度范围为 3% 至 15%。 甘油可能会限制非特异性聚合，如果您计划在数据收集期间使用冷冻技术，它可能会很有用。

在蒸汽扩散结晶实验中添加甘油时，请确保容器中的甘油浓度高于液滴，否则您可能会发现液滴会变得更大而不是更小。 参考： Vera,L.、Czarny, B.、Georgiadis, D.、Dive, V.、Stura, E.A. (2011) 甘油在蛋白质结晶中的实际应用。 水晶。 成长与发展。 11:2755–2762

What additive to use  蛋白结晶用用什么添加剂

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What additive to use  蛋白结晶用用什么添加剂

When trying to decide which additive might be useful during crystallization, try the following. If one has crystals and wants to try using additives to improve the crystal size or quality go back to the initial crystallization screening plates. Review the plates, looking for conditions where no precipitate nor crystals were/are observed. Now, review the results, looking for a common reagent ingredient present where no precipitant or crystals are found. For example, one might find that all drops with isopropanol remained clear. One could then try adding isopropanol to the current crystallization condition to see if the isopropanol could improve the crystal size and/or quality. If one observes a difference in the crystal in the presence of isopropanol, then one might consider evaluating other additives in the class of alcohols such as ethanol, methanol, tert-butanol and others. If one has no crystals, but plenty of precipitate, phase separation and clear drops, follow the above analysis and try adding the common reagent ingredient found in clear drop to those drops which contained precipitate or phase separation. It is possible this agent could improve or at least manipulate sample solubility. The above tip was submitted from Jarmila Jancarik from the laboratory of Sung Ho Kim at the University of California Berkeley. Thank you Jaru! When using this approach it might be reasonable to discern between concentration independent and dependent precipitation when trying to decide which agent to pursue as an additive. Try evaluating the concentration independent agents first and then look at the other agents if sample quantity permits. For example, if one observe precipitate in 15 to 40% PEG but not in 5 to 10% PEG, it might simply be a concentration. However, if one observes no precipitate in 45 to 60% v/v MPD one could guess that MPD is a reasonable agent to evaluate as an additive.

当试图决定在结晶过程中哪种添加剂可能有用时，请尝试以下方法。如果有晶体并想尝试使用添加剂来改善晶体尺寸或质量，请返回最初的结晶筛选板。检查板，寻找没有观察到沉淀或晶体的条件。现在，查看结果，寻找没有发现沉淀剂或晶体的常见试剂成分。例如，人们可能会发现所有含有异丙醇的液滴都保持透明。然后可以尝试将异丙醇添加到当前的结晶条件中，以查看异丙醇是否可以改善晶体尺寸和/或质量。如果在异丙醇存在下观察到晶体的差异，那么可以考虑评估醇类中的其他添加剂，例如乙醇、甲醇、叔丁醇等。如果没有晶体，但有大量的沉淀、相分离和透明液滴，请按照上述分析，尝试将透明液滴中常见的试剂成分添加到含有沉淀或相分离的液滴中。该试剂有可能改善或至少控制样品溶解度。上述提示由加州大学伯克利分校 Sung Ho Kim 实验室的 Jarmila Jancarik 提交。谢谢雅鲁！当使用这种方法时，在尝试决定使用哪种试剂作为添加剂时，区分与浓度无关的沉淀和依赖沉淀可能是合理的。尝试先评估与浓度无关的试剂，然后在样本数量允许的情况下查看其他试剂。例如，如果观察到 15% 到 40% PEG 中的沉淀，但 5% 到 10% PEG 中没有，则它可能只是一种浓度。然而，如果在 45 至 60% v/v MPD 中没有观察到沉淀，则可以猜测 MPD 是一种可以作为添加剂进行评估的合理试剂。