Re: [ccp4bb] Skin on drops
Gyan, With the addition of DTT to remove the skin, did you see an increase in the resolution with the skin no longer present compared to with it still there? -Adam > On Feb 25, 2015, at 11:15 AM, Gyanendra Kumar wrote: > > Adding DTT in your protein buffer or crystallization solution may also help. > You could try increasing amounts of DTT/BME/TCEP in your crystallization > solution and find a balance between reduction of skin formation vs getting > crystals. > > Adding 2mM DTT in my protein buffer helped me get rid of much of the skin on > the drop in which the crystals were tightly embedded. > > -Gyan > >> On Wed, Feb 25, 2015 at 3:00 AM, Han Remaut wrote: >> Dear Ulrike, >> >> you could try avoid the drop-air interface by overlying sitting drops with >> silicone oil or a 50/50 silicon/paraffin oil mixture. Note that this will >> alter the kinetics with which your drops reach equilibrium, and hence may >> alter your ability to get crystals of the protein. Batch crystallization >> under oil is another option of course. >> >> Adding some alcohols (5-10% EtOH, isopropanol) or detergent (0.5 mM LDAO for >> example) in your crystallization conditions may also be something to >> consider. >> >> If none of these work, I'd concentrate on harvesting the crystals from the >> skin. I tend to cut these open from the side, flip over the skin so that one >> has better access to the crystals that generally are associated with the >> inner face of the skin. You can try peel the crystals off the skin, or cut >> out a piece of skin surrounding a crystal. That will not hurt diffraction >> quality of the crystals. >> >> Hope that helps. >> >> Han >> >> >> >> On 25 Feb 2015, at 09:34, Ulrike Demmer wrote: >> >> > Dear crystallographers, >> > >> > I am trying to crystallize a soluble protein which tends to form >> > aggregates. The crystallization condition is 20% PEG 3350 + 0,2 M >> > Na-Formate. During the crstallization process a thick skin is formed on >> > top of the sitting-drops. As well the crystals are buried in precipitate. >> > Before I start harvesting I try to remove the skin but still it is hardly >> > possible to get any crystals out of these drops. >> > >> > Any suggestions how to avoid the formation of skin on crystallization >> > drops ? >> > >> > Cheers, >> > >> > Ulrike >> >> >> Han Remaut, PhD >> Laboratory of Structural & Molecular Microbiology >> VIB / Vrije Universiteit Brussel >> Building E4, Pleinlaan 2 >> 1050 Brussel >> >> han.rem...@vib-vub.be >> tel. +32-2-629 1923 / +32-499 708050 >> http://www.vib.be/en/research/scientists/Pages/Han-Remaut-Lab.aspx > > > > -- > Gyanendra Kumar, PhD > St. Jude Children's Research Hospital, > Department of Structural Biology, > 262, Danny Thomas Place, MS-311 > Memphis, TN 38105 > Phone: 901-595-3839 > Cell: 631-875-9189 > ---
Re: [ccp4bb] cryoprotectant
Hi Reza, 1.0 and 1.25 M citrate are potential cryo solutions. They still had faint ice rings present but were not dominant. It is also difficult to make a solution more concentrated then 1.75 M citrate. Hope this helps. -Adam > On Dec 1, 2014, at 10:59 AM, Reza Khayat wrote: > > Hi, > > Has anyone used citrate as the sole cryoprotectant? If so, > what concentration was needed? > > Best wishes, > Reza > > Reza Khayat, PhD > Assistant Professor > The City College of New York > Department of Chemistry, MR-1135 > 160 Convent Avenue > New York, NY 10031 > Tel. (212) 650-6070 > www.khayatlab.org
Re: [ccp4bb] metal chelation
Thank you everyone for the comments and suggestions. To answer a few questions: -I do not use a treated buffer system. I have just used the nano-pure water. I have looked into Chelex, but before I bought it I wanted to see if you all recommended it. I was trying to avoid this, but it may not be possible now. -the active site does bind metals and is promiscuous in binding, so it is not know if the His tag or active is the source of contamination, but cleavage is not an option for us. The biding of metal is going to be needed for phasing, so good point Tim, hopefully just not in the His site. -thank you Vivoli for the protocol, seems very thorough. Have you had success with it? I anticipate I'll need to go down this road. -Roger, the metals you mentioned (Zn and Fe) are the problem and I expect to have to go to heroic measures to get an apo enzyme . But you did mention easier ways of getting metal substituted. I have some evidence that I can do this. Do you have any other thoughts on this matter? Maybe a reference to something similar (non-apo but could substitute? Thank you all so much for the help and advice. -Adam > On May 19, 2014, at 12:55 PM, Roger Rowlett wrote: > > The answer depends on a number of questions: > What metal ion are you trying to eliminate? > What kind of metal-binding site is involved? > A peripheral or loose binding site? (e.g. surface calcium ions)--these may > respond to chelators > An active site coordinated metal? (e.g., metalloenzyme)--these can be > refractory > Many metalloenzymes are not going to give up their metal to chelators, or > just any chelator, or at all. Denaturation, dialysis, and refolding is an > extreme way of removing metal ions to make apoprotein. Won't work for every > protein. Chelation can be highly specific, that is one chelator may work, > while another, similar one, will not. > Some metal ions are notoriously difficult to eliminate, because they are > adventitious trace contaminants in nearly everything, e.g. zinc and maybe > even iron. (Plastic-ware seems to be often loaded with trace iron, and also > is capable of adsorbing metal ions form solution.) To make apo-enzymes from > zinc proteins, you have to go to heroic efforts to ensure that glassware, > water, buffers, and reagents are zinc-free, especially if you don't have high > (mM) concentrations of protein to work with. > A His-tag is very likely to snag adventitious metals from solution, and can > often mess up metal analysis for metalloproteins by providing "extra" metal. > If this is a problem for your application, you may want to consider removing > the His-tag. > If you are making apoenzyme to get a different metal installed > (metallosubstitution), there are slightly easier ways to do that than going > through the apoenzyme route. > Cheers, > ___ > Roger S. Rowlett > Gordon & Dorothy Kline Professor > Department of Chemistry > Colgate University > 13 Oak Drive > Hamilton, NY 13346 > > tel: (315)-228-7245 > ofc: (315)-228-7395 > fax: (315)-228-7935 > email: rrowl...@colgate.edu >> On 5/19/2014 1:20 PM, SUBSCRIBE CCP4BB Adam Brummett wrote: >> Hello All, >> >> I apologize for the non-crystal related question. I am trying to get a >> fully metal-free apo enzyme. The 6x His construct is consistently purified >> with some metal (20-30%). I have attempted chelating away the metal with up >> to 30 mM EDTA and DFO and then dialyzing it away, but this has shown little >> to no effect. Any thoughts or recommendations would be greatly appreciated. >> Thanks. >> >> Adam >
[ccp4bb] metal chelation
Hello All, I apologize for the non-crystal related question. I am trying to get a fully metal-free apo enzyme. The 6x His construct is consistently purified with some metal (20-30%). I have attempted chelating away the metal with up to 30 mM EDTA and DFO and then dialyzing it away, but this has shown little to no effect. Any thoughts or recommendations would be greatly appreciated. Thanks. Adam
