Juan,
Humidity variation is what vapour diffusion crystallization achieves.
In your list of all possible dehydration methods you would end up
classifying all
vapour diffusion experiments as a case of dehydration. After nucleation,
crystals
continue to grow and the drop continues to become more concentrated in
precipitant.
I agree you are completely right!
The concept of crystal hydration is very complicated:
Crystals are grown in liquid water and often analysed in vitrified water
with a solvent-hydrogen bond network that is different from that in the
liquid state.
What does this mean in terms of hydration?
The use of cryo-protectant alter the solvent hydrogen bonding pattern.
What does this mean in terms of hydration?
VM, the Matthews coefficient, defined as the crystal volume per unit of
protein molecular weight is a a measure of hydration?
So if the VM is the same the hydration is the same?
I agree that all the methods that you mention will affect hydration of the
crystals, but the way that X-ray crystallography is carried out today
cannot avoid it.
"Crystal dehydration" must be defined as an explicit effort to use
methodology designed to alter the hydration of crystals, preferably using
a defined measured and controlled relative humidity value. As you start
to consider badly defined systems, you will also have badly defined
hydration.
Enrico.
On Wed, 16 Jan 2013 14:18:05 +0100, Juan Sanchez-Weatherby
<juan.sanchez-weathe...@diamond.ac.uk> wrote:
Dear all,
From Leonid's reply earlier you can see a problem some of us have been
having for a while now, when looking for literature regarding
dehydration. Most of you that perform dehydration either don't consider
it happening or don't report it in great detail in your publications.
This is only understandable because it isn't the focus of your work and
it only helps you get to where you want to get to.
I'm trying to get an up to date picture of what is out there but I
haven't got the time or eyes to go through everyone's methods to pick
the couple of lines that describe your particular method. I really want
to find out what is being done to be able to give people better advice.
So: Could people out there that think that in their particular projects
dehydration/hydration had an effect send me a ref. or a short
description? (can be done outside the BB to not spam everyone) I will
duly acknowledge everyone!!
By dehydration I mean:
1 Soaking with increasing concentration of precipitants or salts
2 By equilibrating against a new precipitant or salt (by vapour
diffusion or dialysis)
3 By letting the drops dry (controlled or uncontrolled)
4 by using an FMS/HC1/MicroRT or any other gadget
5 By some other magical trick you may have
Thank you all for your help,
Regards
Juan
====================================
Juan Sanchez-Weatherby, PhD
Beamline Scientist - I02
Macromolecular Crystallography Group
Diamond Light Source Ltd
Diamond House DR1.64
Harwell Science and Innovation Campus
RAL, Chilton, Didcot
Oxfordshire
OX11 0DE
United Kingdom
Tel: +44 (0)1235 778661
Mob:+44 (0)7795 641259
Fax:+44 (0)1235 778052
juan.sanchez-weathe...@diamond.ac.uk
http://www.diamond.ac.uk
====================================
-----Original Message-----
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Leonid Sazanov
Sent: 15 January 2013 19:32
To: ccp4bb
Subject: Re: [ccp4bb] crystal dehydration
In case if dehydration needs to be done slowly and under tight control
of all parameters, one possibility is to use micro-dialysis buttons.
We used it for a large membrane protein complex and diffraction improved
from ~7 to 2.7 A. The crystal is fished out and put into mother liquor
solution in the button, sealed with dialysis membrane and the button is
then placed into about 5 mls of mother liquor with slightly higher PEG
concentration. Then you just exchange outside buffer every day or so for
solutions containing higher concentrations of PEG. We went from ~9 to 30
% PEG4000 in about a week. You can easily observe crystal under
microscope and if it cracks - you went too far/too quickly with PEG and
need to use a bit less next time. Also, this method allows you to
control all other components of the dehydrating solution - we needed to
decrease salt concentration at the same time as increasing PEG. You can
also introduce/increase cryo-protectant concentration at the same time.
With these crystals, otherwise excellent dehydration machines already
mentioned did not work, possibly because the process had to be really
slow. The reference is here: http://www.ncbi.nlm.nih.gov/pubmed/21822288
Best wishes.
--
Enrico A. Stura D.Phil. (Oxon) , Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449 Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE
http://www-dsv.cea.fr/en/ibitecs/simopro/ltmb/cristallogenese
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
