Soaking Kinase Crystals with ATP analogues

From: Pascal Egea
Date: 1 February 2012 22:31


Hi ,

To add to the previous comments,

crystallization of GTP or ATP (or their analogues) with their kinase/ A- or G-tpases can depend on a lot of factors that were mentioned (such as packing).

A simple common problem is that ATP solutions should be carefully buffered prior to their use for soaking, people tend to forget about this. A 100 mM ATP solution is pH 3 (probably not good for your protein and it has 3 acidic groups)

For some classes of ATP binding proteins, acidic pH have also been shown to lower chances of successful soaking or co-crystallization.

The crystallization condition is also important. High concentrations of sulphates or phosphates tend to complicate things . Same thing for high concentrations of di or tri carboxylic acids (such as citrate, tartrate or malonate). Sulfates tend to occupy the beta phosphate binding sites and at high concentrations they can outcompete an analogue. 

For first hand experience, I would not assume that all analogues behave the same. Especially between AMPPNP, ATPgammaS and AMPPCP (or their Guanine counterparts). the Cp analogues in our hands tend to have lower affinities. You can always try ADP AlF4 combination or ADP BeF3, if you are not afraid of beryllium .

Hope this helps

Good luck

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Pascal F. Egea, PhD