I wonder if there is a big difference between dehydrating in a drop, where the amount of
mother liquor is essentially unlimited, and dehydrating a mounted crystal in something
like the FMS, where there is only a thin film of ML on the surface. In the latter case,
once the surface fluid is gone, assuming surface tension prevents air from entering the
channels, the tendency for further evaporation will cause reduced hydrostatic pressure in
the channels, and the pressure differential will exert a physical force to shrink the
crystal (and to oppose further evaporation). If soaking in a droplet with salt at high
osmolarity, salt freely enters the channels, so there is no hydrostatic pressure
difference betwene inside and outside. With PEG it would depend whether the PEG can enter
channels, with large PEG and small channels there would be an osmotic pressure gradient to
shrink the crystal. So it would seem that equilibrating at a certain RH in the FMS vs in a
droplet could have very different results. is there any data on this?
Matthew Bowler wrote:
Hi Andre,
a very effective method is the use of a humidity control device. It has the
great
advantage that you can characterize changes that occur and also move straight
to data
collection. There are several HC1 devices in Europe (developed here at the EMBL
and
available at Diamond, BESSY and MaxLab) and at least 1 in the USA - there is
also the FMS.
You can of course also do this in the lab but the disadvantage is that any
change induced
cannot be observed. The relative humidity (RH) that is in equilibrium with your
mother
liquor is 99%, you could think about slowly replacing the reservoir solution
with
increasing salt solutions so as to dehydrate in the drop - this avoids handling
the
crystal - equations to convert between PEG concentrations and salt
concentrations for RH
matching can be found here:
http://www.esrf.eu/UsersAndScience/Experiments/MX/How_to_use_our_beamlines/forms/equation-4
Below are some links that might help, best wishes, Matt.
Website for HC1 experiments at the ESRF:
http://www.esrf.eu/UsersAndScience/Experiments/MX/About_our_beamlines/ID14-2/HC1b
Calculation website for mother liquor RH equilibria: http://go.esrf.eu/RH
On 29/10/2013 16:18, Andre Godoy wrote:
Dear all
I'm trying to solve a beautiful large crystal that, unfortunately, doesn't go
further
than 5 A resolution. I believe that in this case, the lack of resolution is due
the high
solvent content (about 66%). Therefore, my next strategy should be the
dehydratation.
Yet, I never (sucessfully) did that. I read different approachs, were people
equilibrate
crystals in dehydratation solution for days, or do more than 20 steps, or add
solvents.
Since i never had sucess in my trials, I was thinking that someone can suggest a
protocol (should I remove all salt?, should I keep the additive concentration?,
how much
precipitant should I add? how many steps?).
crystal condition: 23% PEG 3350, 0.2M NaCl, 0.1M Tris pH 8.5, 3% galactose
(orthorhombic
crystals, with about 0.6 x 0.6 mm)
all the best,
Andre Godoy
--
Matthew Bowler
Synchrotron Science Group
European Molecular Biology Laboratory
BP 181, 6 rue Jules Horowitz
38042 Grenoble Cedex 9
France
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