 |
 |
I think one may need to distinguish between three different kinds of dehydration experiment, because of the different forces they will exert on a crystal to shrink the unit cell, creating new stabilizing crystal contacts or perhaps causing contacts to fail in a chaotic manner, disordering the crystal: 1. dehydration while bathed with copious solution, by increasing concentration of glycerol or other small solute. 2. same, but the dehydrating solute is too large to enter (some) solvent channels. Large PEG molecules may do this(?). But PEG is often seen in crystal structures. 3. The crystal is fished out of solution before dehydration, with a thin layer of solution adhering to the surface. This is what is done with the FMS and I suppose other humidity-controlling systems, or more cheaply by fishing the crystal in a cryocap with a short pin and storing it screwed into an upright cryovial with 100 ul or so of defined humectant solution in the bottom. #1 would exert no direct forces on the crystal, but internal surfaces might come together to exclude water, which might lead to shrinkage. #2 could exert osmotic force, as water diffuses out of the crystal to the bulk solvent resulting in lower hydrostatic pressure in the solvent channels. Pressure on the surface of the crystal could then cause shrinkage. in #3, dehydration can reduce the volume of solution to the point where it is insufficient to fill the solvent channels. If surface tension prevents air from entering the crystals, atmospheric pressure on the surface of the crystal will promote shrinkage. I think in some cases "annealing" actually works by dehydration #3. We reported such a case in JMB 351, 573-597 (buried in Methods at the end of the paper, and disc p579). We have diffraction images before and after annealing, and not only the resolution improved dramatically but the cell volume decreased by 18%. And the structure showed a new crystal contact was formed. We're pretty sure this is dehydration because of the volume change and because a similar effect on diffraction and cell param could be obtained (with the same batch of crystals or a few other batches, in other cases something else was limiting and the improvement was not so significant) by simply holding the crystal in the air for 60-90 sec before plunging in LN2. It seems odd that annealing would cause dehydration- you would expect massive condensation on the crystal as it thaws - but I think the heat capacity of the crystal and the amount of ice (if any) to be melted is so small that it reaches room temp before much condensation occurs. Then the down-draft caused by the cold copper pin (sitting vertical with crystal upward) drew enough warm dry air over it to dehydrate. Juan Sanchez-Weatherby 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 > > [log in to unmask] > > http://www.diamond.ac.uk > ==================================== > > -----Original Message----- > From: CCP4 bulletin board [mailto:[log in to unmask]] 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.