Re: [ccp4bb] crystallizing a complex that's sensitive to ionic strength
Have you tried adding water to your reservoir and allowing it to vapor diffuse into the drop? Kris Kris F. Tesh, Ph. D. Department of Biology and Biochemistry University of Houston - Original Message From: Tim Gruene To: CCP4BB@JISCMAIL.AC.UK Sent: Mon, February 28, 2011 3:48:25 AM Subject: Re: [ccp4bb] crystallizing a complex that's sensitive to ionic strength Dear Hua, adding water as suggested by Jan Kern could also be accomplished in a more sophisticated way by using dialysis buttons. They require large volumes, though, 5mul is the minimum as far as I know. At the fancy end of this you could try the TOPAZ system by fluidigm. At the ECM in Darmstadt one of the companies presented a cheap, plasitc based version of this which looked quite appealing to me. Maybe it was the CrystalHarp from Molecular Dimensions, but I am not sure. Cheers, Tim On Sat, Feb 26, 2011 at 09:13:49PM -0500, Hua Yuan wrote: > Dear CCP4 community members, > > I've been trying to crystallize a protein complex that's very sensitive to > ionic strength, i.e., lower salt (~0.3M) will cause precipitation of the > complex but higher salt (~0.5 M) breaks the complex apart. The interaction > that holds the complex is probably mainly ionic type. > The crystals I got so far has only one component of the complex from which > all the crystallization conditions have high salt such as 2M Ammonium > Sulfate in them. Besides repeatly screening many crystallization > conditions, I was wondering whether is any way to work around this problem. > Your suggestions would be greatly appreciated! > > Thanks, > > Hua -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A
Re: [ccp4bb] crystallizing a complex that's sensitive to ionic strength
Dear Hua, adding water as suggested by Jan Kern could also be accomplished in a more sophisticated way by using dialysis buttons. They require large volumes, though, 5mul is the minimum as far as I know. At the fancy end of this you could try the TOPAZ system by fluidigm. At the ECM in Darmstadt one of the companies presented a cheap, plasitc based version of this which looked quite appealing to me. Maybe it was the CrystalHarp from Molecular Dimensions, but I am not sure. Cheers, Tim On Sat, Feb 26, 2011 at 09:13:49PM -0500, Hua Yuan wrote: > Dear CCP4 community members, > > I've been trying to crystallize a protein complex that's very sensitive to > ionic strength, i.e., lower salt (~0.3M) will cause precipitation of the > complex but higher salt (~0.5 M) breaks the complex apart. The interaction > that holds the complex is probably mainly ionic type. > The crystals I got so far has only one component of the complex from which > all the crystallization conditions have high salt such as 2M Ammonium > Sulfate in them. Besides repeatly screening many crystallization > conditions, I was wondering whether is any way to work around this problem. > Your suggestions would be greatly appreciated! > > Thanks, > > Hua -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] crystallizing a complex that's sensitive to ionic strength
Hi Hua, maybe crystallization by reducing ionic strength could work in this case. See for one prominent example the crystallization of Photosystem I by reducing the salt concentration of the mother liquor by slowly adding water (Fromme and Witt, Biochim Biophys Acta, 1365 (1998) 175-184). Greetings, Jan --- Dr. Jan Kern Lawrence Berkeley National Laboratory Physical Biosciences Division One Cyclotron Road, MS 66-326 Berkeley, CA 94720 On Sat, Feb 26, 2011 at 6:13 PM, Hua Yuan wrote: > Dear CCP4 community members, > > I've been trying to crystallize a protein complex that's very sensitive to > ionic strength, i.e., lower salt (~0.3M) will cause precipitation of the > complex but higher salt (~0.5 M) breaks the complex apart. The interaction > that holds the complex is probably mainly ionic type. > The crystals I got so far has only one component of the complex from which > all the crystallization conditions have high salt such as 2M Ammonium > Sulfate in them. Besides repeatly screening many crystallization > conditions, I was wondering whether is any way to work around this problem. > Your suggestions would be greatly appreciated! > > Thanks, > > Hua > > >
Re: [ccp4bb] crystallizing a complex that's sensitive to ionic strength
Have you tried to crosslink your complex after purification and see if you can crystallize that ? Glutaraldehyde comes to mind but also DMA, DMS or DMP. Jürgen On Feb 26, 2011, at 9:13 PM, Hua Yuan wrote: Dear CCP4 community members, I've been trying to crystallize a protein complex that's very sensitive to ionic strength, i.e., lower salt (~0.3M) will cause precipitation of the complex but higher salt (~0.5 M) breaks the complex apart. The interaction that holds the complex is probably mainly ionic type. The crystals I got so far has only one component of the complex from which all the crystallization conditions have high salt such as 2M Ammonium Sulfate in them. Besides repeatly screening many crystallization conditions, I was wondering whether is any way to work around this problem. Your suggestions would be greatly appreciated! Thanks, Hua .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/
Re: [ccp4bb] crystallizing a complex that's sensitive to ionic strength
Hua Peter Sun and S. Radaev wrote a paper a few years ago where they showed by data mining that the crystallization conditions of complexes heavily favor PEG rather than salt conditions, so you are not alone. Radaev and Sun, Crystallization of protein-protein complexes J. Appl. Cryst. (2002). 35, 674-676 You could try microseeding *into random screens* using the crystals that you have to make a seed stock. See Acta Cryst. (2007). D63, 550–554 and http://www.douglas.co.uk/mms.htm. This approach has sometimes worked before for complexes, eg seeding crystals of an antigen-Fab complex with crystals of just the Fab. You can use this approach by hand or with a robot (spin the seed stock if you us a non-contact robot). One problem is that you would normally put quite a lot of Amm Sulf into your wells because you would suspend the seed crystals in the reservoir solution where the crystals used to make the seed stock were found. The conventional volumes to use are 0.3 protein + 0.2 reservoir solution + 0.1 seed stock, so typically 1/3 of the precipitant would be Amm Sulf. My colleagues and I recently submitted a manuscript to Crystal Growth and Design where we look in great detail at suspending seed crystals in other precipitants (instead of Amm Sul in this case). We found that this normally works, eg you can usually make a seed stock with PEG instead of say salt conditions. You can predict whether the PEG (or any other solution) will work for suspension by incubating the crystals in PEG for 24 hours. If the crystals look unchanged after that, you can almost certainly make a seed stock from the solution in question. (If the crystals dissolve, shatter or crack, you MAY be able to make a seed stock with the solution.) We used 100% PEG600, although in restrospect I think 30% - 50% PEG 3000 might be better. I can send you a preprint, but the gist of the relevant part is above. For a review of more radical approaches, see Bing Xiao et al., Optimizing Protein Complexes for Crystal Growth. Crystal Growth & Design, Vol. 7, No. 11, 2007 2215 I hope that helps Patrick On Sun, Feb 27, 2011 at 2:24 AM, Hua Yuan wrote: > Dear CCP4 community members, > > I've been trying to crystallize a protein complex that's very sensitive to > ionic strength, i.e., lower salt (~0.3M) will cause precipitation of the > complex but higher salt (~0.5 M) breaks the complex apart. The interaction > that holds the complex is probably mainly ionic type. > The crystals I got so far has only one component of the complex from which > all the crystallization conditions have high salt such as 2M Ammonium > Sulfate in them. Besides repeatly screening many crystallization > conditions, I was wondering whether is any way to work around this problem. > Your suggestions would be greatly appreciated! > > Thanks, > > Hua > > > -- patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] crystallizing a complex that's sensitive to ionic strength
Hi Hua, Does your complex elute as a single peak from a gel filtration column or show a monomodal dynamic light scattering profile? If not, it may be worth looking into the method of complex formation. You can co-express the subunits, or purify each subunits separately, and then mix them at a particular molar ratio to form the complex. Purification with columns such as gel filtration after complex formation may be necessary for crystallization. Sometimes mixing the subunits at a high salt concentration, and then gradually lowering the salt concentration via dialysis may result in a more stable complex. If the subunits are small (e.g. 100-200 amino acid residues), refolding may be an option. Fusing the subunits and expressing them as a single polypeptide is another possible option. I hope this helps. Sincerely, Wataru On 2011/02/27, at 11:13, Hua Yuan wrote: > Dear CCP4 community members, > > I've been trying to crystallize a protein complex that's very sensitive to > ionic strength, i.e., lower salt (~0.3M) will cause precipitation of the > complex but higher salt (~0.5 M) breaks the complex apart. The interaction > that holds the complex is probably mainly ionic type. > The crystals I got so far has only one component of the complex from which > all the crystallization conditions have high salt such as 2M Ammonium Sulfate > in them. Besides repeatly screening many crystallization conditions, I was > wondering whether is any way to work around this problem. Your suggestions > would be greatly appreciated! > > Thanks, > > Hua > >
[ccp4bb] crystallizing a complex that's sensitive to ionic strength
Dear CCP4 community members, I've been trying to crystallize a protein complex that's very sensitive to ionic strength, i.e., lower salt (~0.3M) will cause precipitation of the complex but higher salt (~0.5 M) breaks the complex apart. The interaction that holds the complex is probably mainly ionic type. The crystals I got so far has only one component of the complex from which all the crystallization conditions have high salt such as 2M Ammonium Sulfate in them. Besides repeatly screening many crystallization conditions, I was wondering whether is any way to work around this problem. Your suggestions would be greatly appreciated! Thanks, Hua
