It sounds a bit silly after that nice theoretical discussion, but I would try poking the existing oily blobs with a hair. Since you may be close to xtal conditions, stirring up the equilibrium a bit may help nucleate something. I've seen this work more than once, although usually with things where we'd seen some sign of crystals already. Also, try new drops with lower [PEG]. Good luck! ===================================== Phoebe A. Rice Dept. of Biochemistry & Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp
---- Original message ---- >Date: Thu, 13 Jan 2011 12:11:54 -0500 >From: CCP4 bulletin board <[email protected]> (on behalf of Edward Snell ><[email protected]>) >Subject: Re: [ccp4bb] Phase Separation >To: [email protected] > >Hi Ruben, > >Timing is everything - We are just going through the proofs of a paper >entitled "What's in a drop? Correlating observations and outcomes to guide >macromolecular crystallization experiments" by Luft, Wolfley and Snell to >appear shortly in Crystal Growth and Design. In putting this together we found >a number of useful references related to the phase separation phenomena - >temperature may be a very useful variable to try. To quote the relative >paragraphs and hope we don't start a huge discussion (with references to the >figures in the paper removed); > >".... There are protein-rich and protein-poor liquid phases. Protein >concentrations of 400mg/mL have been measured in the protein-rich phase, a >concentration comparable to that found in crystals. (1) Experimental and >theoretical studies demonstrate the formation of immiscible liquid-liquid >(L-L) phase separation in the metastable region of the phase diagram forms >only where there are short range, and/or highly anisotropic interactions >between protein molecules, with further experimental evidence that >demonstrates this region is connected with conditions for growing crystals. >(2) > >When the temperature of crystallization is near or below the formation >temperature of a metastable, immiscible L-L phase separation, at high levels >of supersaturation, experimental data and numerical simulations support a >two-step, non-classical nucleation process.(2) In this mechanism a >protein-rich liquid phase first forms. Nucleation takes place from this phase >followed by initial growth of the nuclei sometimes into the protein-rich and >other times into the protein-poor environment. Haas and Drenth(2) suggest that >this growth mechanism can lead to fewer crystal defects and more rapid crystal >growth as molecules in the concentrated liquid protein phase that surrounds >the crystal are not driven to the surface of the crystal by diffusion and >therefore misaligned molecules can be more readily exchanged. Literature also >supports that it is not the higher protein concentration within the coacervate >droplets or the molecular fluidity that may initiate nucleation but rather an >interface effect between the dense liquid of high-protein concentration in the >droplet and the immiscible surrounding liquid of low-protein concentration.(3) > >When a L-L phase separation is observed ... if one phase is protein-rich and >the other protein-poor, then the system is very close to conditions that have >the potential to produce crystals. If the protein contains tryptophan >residues, then the presence of a protein-rich phase can be verified using UV >fluorescence, .... Crystals will sometimes form from the dense liquid phase >without intervention; .... As is the case with metastable conditions, this >protein-rich immiscible liquid phase can be used for seeding.(4) The other >useful and effective option to induce crystal formation is to drive the system >towards a higher level of supersaturation, the labile state, using >temperature. The rationale for this approach is to increase the attraction >between protein molecules by decreasing the temperature.(5) However, this >process will be dependent upon the solubility properties of the >protein/solvent. Protein solubility is dictated by the combination of the >protein and its chemical environment. The same protein can have increased >solubility at higher temperatures in one chemical environment, and lower >temperatures in a different chemical environment. If the protein/solvent is >more soluble at higher temperatures and L-L phase separation is seen in the >drop, then decreasing the temperature will drive the system towards a higher >level of supersaturation. The opposite applies in cases where the >protein/solvent exhibits retro-solubility, i.e. the protein is more soluble at >lower temperatures. In this case the experiments would be moved to a higher >temperature environment, or set up at a higher temperature in a replicate >experiment." > >1. Kuznetsov, Y. G.; Malkin, A. J.; McPherson, A. Journal of Crystal >Growth 2001, 232, 30-39. >2. Haas, C.; Drenth, J. Journal of Physical Chemistry B 2000, 104, 368-377. >3. Vekilov, P. G. Crystal Growth & Design 2004, 4, 671-685. >4. Bergfors, T. J Struct Biol 2003, 142, 66-76. >5. Dumetz, A. C.; Chockla, A. M.; Kaler, E. W.; Lenhoff, A. M. Biophys J >2008, 94, 570-583. > >Hope this helps, > >Cheers, > >Eddie > > >Edward Snell Ph.D. >Assistant Prof. Department of Structural Biology, SUNY Buffalo, >Senior Scientist, Hauptman-Woodward Medical Research Institute >700 Ellicott Street, Buffalo, NY 14203-1102 >Phone: (716) 898 8631 Fax: (716) 898 8660 >Skype: eddie.snell Email: [email protected] >Telepathy: 42.2 GHz > >Heisenberg was probably here! > >From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Ruben >Van der Meeren >Sent: Thursday, January 13, 2011 4:56 AM >To: [email protected] >Subject: [ccp4bb] Phase Separation > >Dear all, > >I'm trying to crystallize a small, soluble part of a protein (~15kDa, 152AA). >I did some standard screens (Crystal Screen I & II + Index screen) with a >protein concentration of 25 or 45mg/mL in an 1:1 (0.75µL)96 well set up. In >most of the conditions I got phase separation (mostly PEG conditions)! >Precipitation was formed in conditions with salt. I did not have phase >separation with the control (buffer only, see below). For so far I know my >protein was soluble up to a concentration of 60mg/mL (I didn't went higher). >Its predicted to have a lot of beta-strands (according to CD-spectra and >secondary structure predictions). > >So here are my questions: >- What is the molecular basis of phase separation? I mean what is going on at >molecular level? I would suspect that my protein is not soluble in a PEG >environment, is this correct? >- What can I do to prevent my protein or buffer (?) going into phases? Is it >temperature dependent? Are there additives I can add? Do I need to lower the >salt concentration? >- Are there examples (some of your personal experience) where phase separation >was a good thing? > >For your record: the protein is in a 150mM NaCl, 20mM HEPES pH7.5 buffer and >the pI is 5-6. It is cloned with a his-tag (but cleaving the his-tag didn't >change much). > >Best Regards, >Ruben > >____________________________________________________________________ >Ruben Van der Meeren >Ghent University >L-ProBE, hoogbouw, verdiep 5 >K. L. Ledeganckstraat 35 9000 Ghent (Belgium) >E-Mail: [email protected]
