Re: [ccp4bb] crystals with large solvent content -dehydratation
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/ ===
Re: [ccp4bb] crystals with large solvent content -dehydratation
Hi Andre, Are you sure that the cryoprotectant has been optimized?..I am sure you must have already done it but just in case..sometimes the cryo protectant which makes the ice rings disappear is not the best one and playing around that concentration as well as different alternatives can have a huge impact. Another thing, again trivial, is to shoot different parts of the crystal. For big crystals sometimes the regions near the tips diffract better than regions in the interior ( plausibly correlated with penetration of the cryoprotectant during treatment as well as other deformities in the central portions that might have occurred during crystal growth or vitrification.) My humble two cents, Best, Arka Chakraborty On Tue, Oct 29, 2013 at 5:17 PM, Danilo Belviso wrote: > Dear Andre, > > you could try with the protocol described in the following paper > > Acta Crystallogr D Biol Crystallogr. 2013 69,920-3. > Using high-throughput in situ plate screening to evaluate the effect of > dehydration on protein crystals. > Douangamath A, Aller P, Lukacik P, Sanchez-Weatherby J, Moraes I, > Brandao-Neto J. > > It gives very good results with membrane proteins where the water content > is high. > > Danilo > > > On Tue, 29 Oct 2013 08:18:21 -0700, 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 >> > -- *Arka Chakraborty* *ibmb (Institut de Biologia Molecular de Barcelona)** **BARCELONA, SPAIN** *
Re: [ccp4bb] crystals with large solvent content -dehydratation
Hi, you could try dehydration in microdialysis buttons - this allows for slow gradual increase in PEG over few days and full control of other parameters, including lowering salt concentration. It was the only dehydration method that worked well for our large membrane protein: http://www.ncbi.nlm.nih.gov/pubmed/21822288 Described in more detail here: http://www.ncbi.nlm.nih.gov/pubmed/24059518
Re: [ccp4bb] crystals with large solvent content -dehydratation
Dear Andre, you could try with the protocol described in the following paper Acta Crystallogr D Biol Crystallogr. 2013 69,920-3. Using high-throughput in situ plate screening to evaluate the effect of dehydration on protein crystals. Douangamath A, Aller P, Lukacik P, Sanchez-Weatherby J, Moraes I, Brandao-Neto J. It gives very good results with membrane proteins where the water content is high. Danilo On Tue, 29 Oct 2013 08:18:21 -0700, 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
Re: [ccp4bb] crystals with large solvent content -dehydratation
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/ ===
Re: [ccp4bb] crystals with large solvent content -dehydratation
Dear Andre, We had a similar case where the crystals were bigger and diffraction was lousy. Our standard dehydration approaches were not very successful. I suggest reading this - Post-crystallization treatments for improving diffraction quality of protein crystals. Heras B<http://www.ncbi.nlm.nih.gov/pubmed?term=Heras%20B%5BAuthor%5D&cauthor=true&cauthor_uid=16131749>, Martin JL<http://www.ncbi.nlm.nih.gov/pubmed?term=Martin%20JL%5BAuthor%5D&cauthor=true&cauthor_uid=16131749>.Acta Crystallogr D Biol Crystallogr.<http://www.ncbi.nlm.nih.gov/pubmed/16131749#> 2005 Sep;61(Pt 9):1173-80. Epub 2005 Aug 16. But we were able to get a different crystal form (~2A) with high solvent content (75%) by making a bunch of surface mutants. I am not saying that surface mutagenesis is the last resort but something that you might want to consider. See this paper - http://www.jbc.org/content/early/2012/01/31/jbc.M111.327536 Best Regards Harkewal From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Andre Godoy [andre_go...@yahoo.com.br] Sent: Tuesday, October 29, 2013 10:18 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystals with large solvent content -dehydratation 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
Re: [ccp4bb] crystals with large solvent content -dehydratation
Dear Andre, 66% solvent is on the high side but not a good reason for poor resolution. Other with similaa solvent content have achieved resolutions of 1.5 Ang. and even better. I would screen at a lower protein concentration. It will require more precipitant and you should end up with less water in the cell. Enrico. On Tue, 29 Oct 2013 16:18:21 +0100, 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 -- 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 http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://scholar.google.com/citations?hl=en&user=Kvm06WIoPAsC&pagesize=100&sortby=pubdate http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Re: [ccp4bb] crystals with large solvent content -dehydratation
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/ ===
[ccp4bb] crystals with large solvent content -dehydratation
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
