[ccp4bb] problem in heavy metal soaking
I'm working with a protein which crystallizes in a mixture of PEG6K with 0.2M AmSO4,my question is if there's any problem if I want to soak heavy metal derivatives in this crystallizing condition? Does AmSO4 interfere in heavy-metal soaking ? if yes, what's the reason? Thank you in advance.
Re: [ccp4bb] problem in heavy metal soaking
On 26 Sep 2010, at 9:00 AM, Seema Nath wrote: I'm working with a protein which crystallizes in a mixture of PEG6K with 0.2M AmSO4,my question is if there's any problem if I want to soak heavy metal derivatives in this crystallizing condition? Does AmSO4 interfere in heavy-metal soaking ? if yes, what's the reason? Thank you in advance. There's a nice discussion of heavy atom chemistry and why ammonium ions can be problematic in Greg Petsko's review from the 80's: Methods Enzymol. 1985;114:147-56 The bottom line is that amines (and ammonium is an amine) can participate in coordination chemistry for certain metals. If memory serves some of this is also covered in Blundell and Johnson (? not sure about that, I don't have my copy at hand). Good luck, Pat --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
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[ccp4bb] Regarding quality of protein for crystallization
I am working on 45 kd protein (pI 5.3) for crystallization . It is in pGEX vector.I do the expression (induction 24 degree 16 hrs) and purification from Rosetta 2 DE3 cells (has additional rare codon).when I run the affinity chromatography purified protein (cleaved protein after removal of tag) on SDS PAGE I do find 2 bands (one of 45 kd and other 2-3 kd less). The same pattern being retained after FPLC. Intensity of both the bands remain same even after one or two week of storage at 4 degree. Peptide mass fingerprinting (after trypsin digestion) suggest both are my proteins except minor difference in some peptide peaks. Is it because of rare codon/degradation of protein of interest or any other possibilities? Can I use this mixure for crystallization? With Regards Vikrant
Re: [ccp4bb] Regarding quality of protein for crystallization
What are the minor differences in peptides ? Any PTM's perhaps ? Jürgen - 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/ On Sep 26, 2010, at 3:43 PM, vikrant saa wrote: I am working on 45 kd protein (pI 5.3) for crystallization . It is in pGEX vector.I do the expression (induction 24 degree 16 hrs) and purification from Rosetta 2 DE3 cells (has additional rare codon).when I run the affinity chromatography purified protein (cleaved protein after removal of tag) on SDS PAGE I do find 2 bands (one of 45 kd and other 2-3 kd less). The same pattern being retained after FPLC. Intensity of both the bands remain same even after one or two week of storage at 4 degree. Peptide mass fingerprinting (after trypsin digestion) suggest both are my proteins except minor difference in some peptide peaks. Is it because of rare codon/degradation of protein of interest or any other possibilities? Can I use this mixure for crystallization? With Regards Vikrant
Re: [ccp4bb] Regarding quality of protein for crystallization
I assume that the loss of the peptides that you observe for the smaller protein is at the other terminus from the tag? If it is at the terminus where the tag was it would suggest that removal of the tag using proteolysis is the most likely cause. Though saying that Factor Xa/thrombin is not 100% accurate and still maybe causing the slight degradation. You do not say if you see the two different species before cleavage. If you do, have you tried adding protease cocktail inhibitors before disruption of the cells? As you know which peptides are missing, you know roughly the region that is deleted. Does this cause a change in the pI of the smaller protein? If so you may try ion exchange chromatography to separate the two species. For crystallography purposes, two different species may effect crystallization through disruption of crystal packing, though in situ proteolysis crystallization can generate a mixture of different species with one of the fragments being more readily crystallizable. There is only one way to find out... Daniel A. Bonsor, Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472 USA
[ccp4bb] Home Source Options
I'm interested in opinions/advice on home source systems. As synchrotron beamlines are more powerful and more accessible than ever, a home source is really only needed for crystal screening, if at all. With that idea in mind, what are options out there for buying and running a home source in the most cost friendly way possible? I'm aware of most of the options from the big players (Rigaku, Oxford, Bruker), but I would like input on which setups cost the least to buy, run year in and year out, and require the least in terms of facilities setup (cooling water, power supply, etc.) Any positive/negative experiences worth sharing? Thanks, --Paul
Re: [ccp4bb] problem in heavy metal soaking
Hi Seema, In theory, Ammonium Sulfate contains some small fraction of neutral ammonia which can act as a strong nucleophile and react with many heavy metal compounds. That being said, I recently phased two structures with mercury soaks, both of which contained ammonium sulfate. The first was a thimerisol soak with 2M AmSO4 in the mother liquor, and the second with a MeHgCl soak with 0.2M AmSO4. Both were fairly routine. I think others will agree that with heavy metals, logic and theory can go right out the window. There is no way of knowing if it will work, you just have to try. One tip I can offer is to use fresh stocks of metals dissolved at saturating concentrations in water and used on the same day. The fresher the better in my experience, but it could just be voodoo. Good luck! --Paul --- On Sun, 9/26/10, Seema Nath seema.n...@saha.ac.in wrote: From: Seema Nath seema.n...@saha.ac.in Subject: [ccp4bb] problem in heavy metal soaking To: CCP4BB@JISCMAIL.AC.UK Date: Sunday, September 26, 2010, 9:00 AM I'm working with a protein which crystallizes in a mixture of PEG6K with 0.2M AmSO4,my question is if there's any problem if I want to soak heavy metal derivatives in this crystallizing condition? Does AmSO4 interfere in heavy-metal soaking ? if yes, what's the reason? Thank you in advance.
Re: [ccp4bb] Regarding quality of protein for crystallization
1. your information is helpful but not enough to tell if this double-band pattern is a product of e.g. proteolysis or of abortive translation. You don't specify what these 'minor differences' are - in the world of MS there is no such thing as 'minor' since every difference is either a significant one, or experimental error (which for the peptide MS range should be well below 1Da). 2. the range of possibilities is rather large. For starters, since you're not measuring the full-size m.w. (or if you are, you're not telling us) who says that your *lower* band is not your 'correct' protein and in fact the higher '45 kDa' band is not some sort of post-translational modification resulting in an *apparently larger* m.w. on SDS-PAGE. Furthermore, in some really odd cases proteins can give double bands on a gel even without extensive PTM: such as if you have a partially reduced S-S bond in your protein that runs on a gel in an incompletely denatured form. 3. the presence of two bands (even if one is a contaminant) is by no means a deterrent to successful crystallization. Just a few weeks ago my lab crystallized a protein sample that has two major bands in it, about 12 kDa apart - that was an unavoidable outcome of preparative proteolysis, performed on the protein in order to get it to crystallize. Diffraction was fine, structure was solved, and most importantly the protein was found to be correct -- which, if you have multiple bands, is not always the case. We all can tell you various funny/scary anecdotes about folks crystallizing the wrong protein out of a mixture (but Mom, it looked *pure* on the gel, like totally!) Good luck, Artem I am working on 45 kd protein (pI 5.3) for crystallization . It is in pGEX vector.I do the expression (induction 24 degree 16 hrs) and purification from Rosetta 2 DE3 cells (has additional rare codon).when I run the affinity chromatography purified protein (cleaved protein after removal of tag) on SDS PAGE I do find 2 bands (one of 45 kd and other 2-3 kd less). The same pattern being retained after FPLC. Intensity of both the bands remain same even after one or two week of storage at 4 degree. Peptide mass fingerprinting (after trypsin digestion) suggest both are my proteins except minor difference in some peptide peaks. Is it because of rare codon/degradation of protein of interest or any other possibilities? Can I use this mixure for crystallization? ** With Regards *Vikrant *** **
