While there is no systematic study (I think) on this we have observed RH
control systems and concentration of solutes can have the same effect -
Photosystem 1 crystals were dehydrated by transferring them from 20% to
40% PEG6000 resulting in a smaller unit cell and better diffraction
properties - this is a decrease in the vapour pressure above these
solutions from 99 to 96.5% RH. We found that we could reproduce the
same transition using the HC1 from 99 to 97% in RH. This also applies
to cases where glycerol and ethylene glycol have been used. I agree with
your assessment that there is a difference between osmotic pressure and
hydrostatic pressure but it does seem to depend more on the mole
fraction of water in the system - this is directly proportional to the
vapour pressure above a solution (Raoult's law). I have always presumed
that by removing water molecules in the solvent channels (either by
reducing RH surrounding xtals or by replacing them with something else
in the channels) will 'exert pressure' on the crystal lattice -
unfortunately I have no evidence for this, best wishes, Matt.
On 29/10/2013 17:13, Edward A. Berry wrote:
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
===================================================
Tel: +33 (0) 4.76.20.76.37
Fax: +33 (0) 4.76.88.29.04
http://www.embl.fr/
===================================================
--
Matthew Bowler
Synchrotron Science Group
European Molecular Biology Laboratory
BP 181, 6 rue Jules Horowitz
38042 Grenoble Cedex 9
France
===================================================
Tel: +33 (0) 4.76.20.76.37
Fax: +33 (0) 4.76.88.29.04
http://www.embl.fr/
===================================================
