Soaking Kinase Crystals with ATP analogues

From: Dianfan Li
Date: 6 February 2012 09:49

Dear all,

Thanks a lot for good suggestions on this topic. To answer some of the questions: The apo strucuture has not been solved; the crystals dissolved upon soaking (my term 'crack' was not accurate); and I have not tried co-crystallization.

A brief summerization here:

1) Soak/Co-crystallization with ADP + Aluminium fluoride/beryllium fluorid (Thank you Gregory and Pascal) or just ADP (Thanks Sofia).

2) Try other ATP analogues (ATP gammaS, AMP PCP). (Thank you Nat)

3) Soak the crystals with lower concentration, shorter time, slow addition (Thank you Thierry, Artem and Francis)

4) Try other crystal forms if available. I actually tried two other crystal forms and one of them survived soaking overnight. If the analogues got there this would be really good. (Thanks Boaz!)

5) Pay attention to the mother liqour used in soaking. Cryo protectant could help (thanks Pius); High sulphate, phosphate, di/tri-carboxylic acid can be a problem; high concentration of ATP can acidify the mother liquor (thanks Pascal).

6) Try inhibitors (Thanks Alex but I do not have any yet)

7) On the protein side, try mutant that does not hydrolyze ATP (I do have one mutant but was not giving crystal. Cheers Filiz)

8) Cross-linking the crystals using glutaraldehyde before soaking (very good point Ed).

Best regards,

Dianfan