Loss of a zinc at a dimer interface, triggered by formation of a disulfide bridge across the interface has already been observed for nitric oxide synthase. The 1st reported structure had the disulfide bridge: http://www.sciencemag.org/cgi/content/abstract/279/5359/2121
But 2 other groups independently found a structural zinc coordinated by 4 cys, 2 from each monomer: http://www.cell.com/content/article/abstract?uid=PIIS0092867400817183 and: http://www.nature.com/nsmb/journal/v6/n3/abs/nsb0399_233.html Since the protein still has the zinc prior to the crystallization trial, I suggest to try to limit as much as possible the amount of oxygen in your buffer. You could degas the precipitant (preferentially at room temperature), then bubble an inert gas (nitrogen is cheap ...) in (preferentially in the cold, even if you need to let the buffer re-equilibrate at the temperature of your crystallization conditions. The rationale for using two temperatures is that gas solubility decreases with temperature). The trail can also be moved in a small plastic bag connected to a nitrogen line, and punctured with a small hole to maintain a constant nitrogen flow. This very cheap set-up works very well as an economical anaerobic chamber (we've had success with an oxygen-sensitive protein using this set-up). The only oxygen left will be in the air initially trapped between the cover slip and the well solution. But most plastics let oxygen diffuse through: as long as you have enough initial reducing agent to protect the protein for ~ 24 hrs, the above should work. Good luck Thierry -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Engin Ozkan Sent: Thursday, September 25, 2008 06:58 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] losing zinc during crystallization Here is my two cents... How strong zinc is captured by the protein is very protein dependent: I always thought that a great case for this variability was the protection of RING ubiquitin ligases against NEM. Cysteine-catalyzing HECT ubiquitin ligase are killed by NEM; RING finger ligases, where zinc is assumed to be only structural, are thought to be NEM resistant... Well, until you actually do the experiment and see that some RING ligases are also inactivated by NEM! My theory was that I had a case of weak zinc coordination, where oxidation of the zinc-coordinating cysteines was a big issue. DTT made it worse, TCEP kept the protein unaggregated the longest. That's just one protein, though. Engin Artem Evdokimov wrote: > > Please note that TCEP decomposes and one of the decomposition products > is phosphate. Enough TCEP and you might have Zinc Phosphate crystals > which can sometimes look very odd and 'protein looking'. > > Artem > > ------------------------------------------------------------------------ > > *From:* CCP4 bulletin board [mailto:[EMAIL PROTECTED] *On Behalf > Of *Jennifer Han-Chun Tsai > *Sent:* Thursday, September 25, 2008 6:18 PM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* Re: [ccp4bb] losing zinc during crystallization > > I don't know if anyone had experience of TCEP inducing zinc acetate to > precipitate. This paper mentioned this. > > The Crystal Structure of the Olfactory Marker Protein at 2.3 Å Resolution > > *Paul C. Smith, Stuart Firestein and John F. Hunt* > > *Journal of Molecular Biology > <http://www.sciencedirect.com/science/journal/00222836> > Volume 319, Issue 3 > <http://www.sciencedirect.com/science?_ob=PublicationURL&_tockey=%23TOC%236899%232002%23996809996%23319471%23FLA%23&_cdi=6899&_pubType=J&view=c&_auth=y&_acct=C000049198&_version=1&_urlVersion=0&_userid=952835&md5=2d1212952cf958e128dad1358783edf4>, > > 7 June 2002, Pages 807-821 * > > I saw if protein sample containing TCEP would either form > precipitation or weird and rounded shape of small molecule crystal but > not salt. I have tried to crush that rounded shape crystal. It's quite > soft, very similar to the way protein crystals got crushed. > > Does anyone have this experience? Or actually this kind precipitation > could get nice crystal after optimization? > > Thanks, > > Jennifer Tsai > > On Thu, Sep 25, 2008 at 4:53 PM, Eric <[EMAIL PROTECTED] > <mailto:[EMAIL PROTECTED]>> wrote: > > We had a zinc-finger containing protein that we were soaking with > different heavy atom compounds. It turns out KAu(CN)2 provided the > best diffraction of several soaks. We found out it was because the > gold had replaced the zinc ion and was coordinated by the Cys and > His's. The lab nickednamed the protein Goldfinger! Although we didn't > have the problem with disulfides, perhaps a similar gold soak might > help if you tried to crystallize the protein in the presence of TCEP > as well. > > HTH > > ----- Original Message ----- From: "Sue Roberts" > <[EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]>> > To: <CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>> > Sent: Thursday, September 25, 2008 4:35 PM > Subject: losing zinc during crystallization > > > > Hello Everyone > > I've been trying to crystallize a zinc-containing enzyme for what > seems to me to be an eternity. The protein contains stoichiometric > zinc (1 zinc/ protein monomer) when isolated and the zinc is required > for activity. Each crystal we've obtained has lost the zinc and > contains a disulfide bond between two cysteine residues that should be > zinc ligands (based on structures of similar proteins). > > We've tried crystalizing in the presence of reducing agents, > crystallizing with substrate analogs, and supplementing the > crystallization drops with zinc with no success (and combinations of > these approaches). We've obtained a variety of crystals and > determined structures, but none contain any zinc. > > Attempts to insert zinc into the crystal (zinc + reducting agent or > zinc alone) have not been successful. > > Does anyone have any tricks to suggest that might help? > > Thanks in advance. > > Sue > > Dr. Sue A. Roberts > Biochemistry & Molecular Biophysics > University of Arizona > 520 621 8171 > [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]> > ********************************************************************* This message and any attachments are solely for the intended recipient. 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