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sajid akthar wrote: > 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/ > > > ------------------------------------------------------------------------ > > Here are a couple of suggestions: 1. To improve crystal from, you might try some additives, e.g., low concentrations (10-100 mM for solids, 1-5% for liquids) of ammonium sulfate, ethylene glycol, DMSO, lithium sulfate, magnesium chloride, dioxane, sodium potassium tartrate, sodium acetate, calcium chloride, etc. Occasionally additives can significantly improve crystal form. We recently successfully improved the crystallization form of some very challenging needles or plate stacks of a protein by adding 100 mM NaOAc or sodium potassium tartrate. 2. If mosaicity is an issue, it may arise or be exacerbated by the cryopreservation step. Evaporation of drops during handling is frequently a problem in crystal degradation during preparation for freezing. For proteins that do not tolerate glycerol well, we have found an equivalent (or slightly smaller) concentration of glucose is just as effective, but normally less disruptive of crystals. A very gentle way of introducing cryoprotectant is to pipet mother-liquor + cryopreservative directly into the crystal drop a little bit at a time, allowing time for mixing while inverted over the well for a few minutes in between additions. We use a protocol where we add 0.25, 0.25, 0.50, 1.0, and 2.0 drop volumes of the added solution. This is an extraordinarly gentle way to introduce cryoprotectants, and we have yet to observe crystal cracking with this method, although it may increase mosaicity. 3. Alternatively, if your crystals will form at low temperature (4 deg C) you can transfer and cryosoak a crystal for just a few seconds at this temperature prior to collection and immersion in liquid nitrogen. Evaporation of the drop is greatly reduced at 4 deg C, and it is possible to freeze otherwise very finicky crystals this way. Cheers, -- ------------------------------------------------------------------------ Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [log in to unmask]