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Dear Enrico, I agree that in broad terms even bad weather (happens a lot here) could be considered a change in humidity. I'm not trying to claim that every time someone changes something in their drop dehydration is to be blamed. But it is a fact that by simply opening your drop you are causing a change and that change may be the difference between good and bad diffraction. I'm trying to capture people's experiments where they know for a fact that something they did (that could be attributed to a change in humidity) made the difference between having the dataset they wanted or not. The way I've meant to understand hydration is double. Firstly the very clear case where deliberately the sample has been dried and then allowed to hydrate again or where the sample has been hydrated above it starting state. It has been documented a couple of times with the FMS and I think at least once with the HC1. Secondly the case where (and it happens) people soak/cryo-protect) in a solution that has less osmotic power (and lower measurable RH) than that of the original solution. It is done very frequently when for example people add well solution to their nice crystal (and protein and ML where quite different) or add a less dehydrating cryo-protectant solution than the sample had in the first place. And yes cryo-protection by its very nature is a dehydration process it mainly works by hydrogen bonding water so it isn't available for nucleation. Another matter is weather a measurable effect can be linked to the process done to the samples. For example, you can dehydrate lysozyme (to about 70%) and not see any difference in lattice parameter, mosaicity, etc (but it is very dehydrated). VM and any other measure is just the physical consequence of a lattice shrinking where your solvent/protein ratio is changing. The key is weather the change is useful or not. For example photosystem I or II (can't remember exactly) had a very clear pattern correlating approximate lattice parameter with resolution limit in deposited structures. As resolution improved lattice parameters where smaller. Coincidently they also show an improvement with controlled dehydration. I'm not looking for anecdotes I am looking for cases where people are confident that process A gave them something and process B gave them something better and that they might have also observed a lattice change, space group shift, etc. I know there are lots of good cases out there but I can't get hold of them. I hope this is clearer, Juan ==================================== Juan Sanchez-Weatherby, PhD Beamline Scientist - I02 Tel: +44 (0)1235 778661 Mob:+44 (0)7795 641259 ==================================== -----Original Message----- From: Enrico Stura [mailto:[log in to unmask]] Sent: 16 January 2013 13:51 To: ccp4bb; Sanchez-Weatherby, Juan (DLSLtd,RAL,DIA) Subject: Re: [ccp4bb] side question re crystal dehydration 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 <[log in to unmask]> 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. -- 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: [log in to unmask] Fax: 33 (0)1 69 08 90 71 -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. 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