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/
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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: [EMAIL PROTECTED]
