Indeed, if Phoebe Rice is right, and you have dimethyl arsenic bound to your cysteines, you may have a homogeneity problem, not from the protein sample, but from the chemical reaction which is occurring prior to crystallization.
If you have DTT, check that it is fresh. Make sure your cacodylate is fresh also, since old stock may not react so well (yes, this happened to me). Essentially the arsenic and DTT must react to form a reactive species which then reacts with the cysteines. Perhaps you will notice anomalous scattering with even with "native" crystals around the arsenic edge, and perhaps also you can exploit this in phasing. http://skuld.bmsc.washington.edu/scatter/data/As.html Good luck, Mark P.S. Here's a reference for the reaction of cacodylate with cysteines. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WK7-45M7T0C-1B&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=83f1ce7ecb1c07b7d0202220f556cf4e 2008/6/3 Phoebe Rice <[EMAIL PROTECTED]>: > If you have high [DTT] in your buffer, you might be > catalyzing the addition of dimethyl arsenic (from the > cacodylate) to some of your cysteines? > Also, 10% glyercol sounds quite low for reproducibly good > freezing (at least in my experience). > Phoebe > > ---- Original message ---- > >Date: Mon, 2 Jun 2008 17:04:38 +0100 > >From: sajid akthar <[EMAIL PROTECTED]> > >Subject: [ccp4bb] crystallisation > >To: [email protected] > > > > > >Dear All > > > >My protein size is ~30kD and crystallizes with > >19%Peg3350, 0.2M Nacl, and 0.1M Na Cacodylate buffer. > > > >Please refer the attached crystal image with this. The > >crystal looks like good enough for home source. These > >crystals appears in 4-5 days at room temp. > > > >Sometimes I'm getting crystals like this, but very few > >in 24 well tray. Most of the time, I found the drop > >contains needles. If I reduce the precipitant little > >bit, I wont find any change in the drop even after > >long time. Changing pH (or temp)of the buffer does not > >help me any better. The crystal appears only around > >5.5pH. > > > >The problem is mosaicity. This crystal diffracted in > >home source upto 3.2A and the mosaicity is 2.5degree. > >Almost all the good crystal like this having same > >mosaicity. > > > >Good cryo condition so far that I found was > >10%Glycerol with mother liquor. Other conditions > >weekens the diffraction quality or increase mosaicity. > > > >In many crystal I could see some crack in the middle > >of the crystal, it looks like twin crystal. Or the > >crystal appears with some sattelite crystals. > > > >Can anyone suggest me some good way to overcome these > >problems. > > > >Thankz > > > >Sajid > > > > > > > > From Chandigarh to Chennai - find friends all over > India. Go to http://in.promos.yahoo.com/groups/citygroups/ > >________________ > >DSCN0003.JPG (720k bytes) > Phoebe A. Rice > Assoc. Prof., 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 > > RNA is really nifty > DNA is over fifty > We have put them > both in one book > Please do take a > really good look > http://www.rsc.org/shop/books/2008/9780854042722.asp > -- Mark BROOKS Telephone: 0169157968 Fax: 0169853715 Institut de Biochmie et de Biophysique Moleculaire et Cellulaire UMR8619 - Bât 430 - Université de Paris-Sud 91405 Orsay CEDEX Skype: markabrooks
