Dianfan Some kinases have such conformation in non-activated apo form that the ATP binding site is partially obstructed. Soaking an ATP analog may then have 3 outcomes: 1) successfully open up the binding site without damage to the crystal, 2) fail to open up the active site and the compound cannot diffuse to the active site, or 3) induce conformational changes which lead to serious disorder in the crystals (which then loose their diffraction) or even crack.
Hence my question: is the ATP binding site unoccluded in the apo structure? If you're in situation #3 then soaks at low concentrations may get you to #1 as a more "gentle" diffusion may be better accommodated by a crystal. Or you may stay in #3, or you may have lowered the concentration so much that the crystals don't crack and you're end up in situation #2. Still a worthwhile experiment. If the ATP binding site is unoccluded then another possibility would be that the kinase-ATP analog may be more soluble than the apo kinase, in which case increasing the precipitant concentration in your soaking buffer may help. Good luck Thierry -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dianfan Li Sent: Wednesday, February 01, 2012 2:17 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Soaking Kinase Crystals with ATP analogues Dear all, Sorry about a non-crystallographic question here. I am working on a kinase and would like to get an ATP analogue into the crystals. When soaked with AMP-PCP, the kinase crystals crack in about 15 min at 4 C. I could try other analogues like AMP-PNP etc, but those would probably behavour in a same way as AMP-PCP. Is it a good idea of trying quick soaks at high concentrations of AMP-PCP? Co-crystallization is another option I have but AMP-PCP is a substrate of the kinase (with low rate). What are other ways of getting ATP analogues into a crystal? Thanks for suggestions, Dianfan Dianfan Li, PhD College of Biochemistry and Immunology Trinity College Dublin Dublin, Ireland. Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
