[ccp4bb] side question re crystal dehydration
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.
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 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 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/ibitecs/simopro/ltmb/cristallogenese
Re: [ccp4bb] side question re crystal dehydration
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 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
Re: [ccp4bb] side question re crystal dehydration
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:est...@cea.fr] 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 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
