Since last few months I am trying to improve my 40kd protein crystals for which I am getting either needles or multinucleation. I have tried various conditions with varying pH, Temperature, precipitant, protein concentration, sitting drop, hanging drop, under oil, batch method, seeding. So please suggest me how to improve crystals
Regards
You could try growing your crystals in a silica hydrogel matrix. I used this method to reduce the nucleation of a couple of unrelated proteins I was working on a few years ago. I was getting showers of needles and for one of the proteins the gel reduced the number of needles by half and produced slightly fatter needles too. The gel didn't work with the other protein (I found that the addition of glycerol worked for the second protein, glycerol is a well-known nucleation inhibitor). The hydrogel basically works like gelatine in jello. It turns your crystallisation drops into gels and reduces the nucleation process in a physical way. As these gel-forming compounds are inert, they don't affect the chemistry in the drop.
Another option is to try additive screens that tests many different compounds known to enhance/change crystal morphology (e.g. alcohols and particularly dioxane are known to inhibit/reduce nucleation, "poison" crystal surfaces and change the space group of crystals).
Both the gel and the screens are available from Hampton.
You could try growing your crystals in a silica hydrogel matrix. I used this method to reduce the nucleation of a couple of unrelated proteins I was working on a few years ago. I was getting showers of needles and for one of the proteins the gel reduced the number of needles by half and produced slightly fatter needles too. The gel didn't work with the other protein (I found that the addition of glycerol worked for the second protein, glycerol is a well-known nucleation inhibitor). The hydrogel basically works like gelatine in jello. It turns your crystallisation drops into gels and reduces the nucleation process in a physical way. As these gel-forming compounds are inert, they don't affect the chemistry in the drop.
Another option is to try additive screens that tests many different compounds known to enhance/change crystal morphology (e.g. alcohols and particularly dioxane are known to inhibit/reduce nucleation, "poison" crystal surfaces and change the space group of crystals).
Both the gel and the screens are available from Hampton.
Two suggestions that might lead to better crystals.
First, have you looked into limited proteolysis? It could be that your current construct is slightly sub-optimal (e.g. regions of disorder at either terminus). Hampton sells two kits of various proteases (http://www.hamptonresearch.com/product_detail.aspx?cid=30&sid=193&pid=593) that can be used to identify proteolytically stable domains/regions of your protein (e.g. those where regions of disorder have been removed). It is possible (even probable) that these new constructs would produce "better" crystals.
Second, you could examine reductive methylation of surface exposed primary amines (Lysine). This technique (http://www.nature.com/protocolexchange/protocols/250) is extremely easy to use, and has been demonstrated to increase crystallization (http://www.sciencedirect.com/science/article/pii/S0969212606003911) by promoting both intra and inter molecular interactions involving Lysine, which typically is extremely mobile in the native state. In my experience, the highest chance of success with this approach are situations precisely like yours, when crystals show up, but they aren't "good enough".
You can try reducing protein concentration and adding 1-5% glycerol.
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