[ccp4bb] Soaking Kinase Crystals with ATP analogues
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
Re: [ccp4bb] Soaking Kinase Crystals with ATP analogues
Consider cross-linking crystals with glutaraldehyde. The caveat here is that you may end up with the protein conformation that is forced by lattice, but if the issue is just the fragility, you should be fine. I assume that crystals simply crack but do not dissolve? Certainly, as others have said, slower inhibitor addition should be tried. On Wed, 2012-02-01 at 19:17 +, Dianfan Li wrote: 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. -- Hurry up before we all come back to our senses! Julian, King of Lemurs
[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.
Re: [ccp4bb] Soaking Kinase Crystals with ATP analogues
On Wed, Feb 1, 2012 at 11:17 AM, Dianfan Li l...@tcd.ie wrote: 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. This isn't too surprising; most kinases undergo global conformational changes (domain closure) when binding ATP. 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? I'd recommend trying ATP-gammaS - it could also be a substrate, but it's worth a look. (Is there any reason to believe that AMP-PNP is a substrate?) I've noticed that the various analogues have been known to result in different conformations in the crystal structure, so it may be a good idea to try more than one anyway. -Nat
Re: [ccp4bb] Soaking Kinase Crystals with ATP analogues
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.
Re: [ccp4bb] Soaking Kinase Crystals with ATP analogues
On Feb 1, 2012, at 12:17 PM, Dianfan Li wrote: 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. 15 minutes is a long time. Scoop crystals during that time period. Do the cracked crystals diffract? Do you see the analogue? F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Soaking Kinase Crystals with ATP analogues
I assume that cocrystallization has failed? What you are experiencing is likely the effect of conformational transition caused by ligand. You can try very slow adition (even microdialysis) or if your ligand is fairly insoluble then you can just add a tiny solid particle of inhibitor to your drop and hope it dissolves slowly and saturates the crystal slowly. Hope it helps :-) Artem On Feb 1, 2012 1:31 PM, Dianfan Li l...@tcd.ie wrote: 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.
Re: [ccp4bb] Soaking Kinase Crystals with ATP analogues
Hi, First, it's very much a crystallographic question. Second, the success or failure in soaking in ligands/cofactors depends quite often also to the crystal packing. Some packing forms (and the spacegroups that go with it) will tolerate the soaking even if it's accompanied with a conformational change, whereas others won't (like the spacegroup you currently have). So you could try, among the other good suggestions that you were given, more crystallization conditions, perhaps you might get lucky and come across crystals in a different spacegroup which will behave more nicely vis-a-vis soaking. Good luck, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Dianfan Li [l...@tcd.ie] Sent: Wednesday, February 01, 2012 9:17 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.
[ccp4bb] Subject: [ccp4bb] Soaking Kinase Crystals with ATP analogues
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 -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] Soaking Kinase Crystals with ATP analogues
Dear Dianfan, In some cases the ATP-lid of the kinase is blocking the active site in the crystal form. In those cases the only option is to try co-crystallisation. Besides ATP and the homologs you mention you can also try ADP that as you will see in the PDB has been heavily used for kinases. Best of luck Sofia Sent via BlackBerry® from Vodafone -Original Message- From: Dianfan Li l...@tcd.ie Sender: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK Date: Wed, 1 Feb 2012 19:17:03 To: CCP4BB@JISCMAIL.AC.UK Reply-To: Dianfan Li l...@tcd.ie 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.
Re: [ccp4bb] Soaking Kinase Crystals with ATP analogues
Maybe someone has suggested this already... If so, I am re-enforcing it. If the cracking is coming from actual molecular movement induced by binding (and not other reason like differing ionic strength in your soaking conditions) you could try setting up some co-crystallization and (hopefully) grow some enzyme-substrate complex crystals... hth yuri
