Re: [ccp4bb] Control the crystallization process in the presence of small volatile organic molecules
Hi Chen, I had a similar condition before (reservoir solution with 10% dioxane) and an oil-based cryoprotectant (66.5% w/w paraton-N, 28.5% paraffin oil and 5% glycerol) worked for me. Please check this paper out: http://scripts.iucr.org/cgi-bin/paper?wd5052 In addition, have you tried cryoprotection without removing the sealing membrane? I did it by punching a hole on the membrane with a needle, taking away the reservoir solution with a syringe, and adding the cryo solution to the reservoir. You can then wash the crystals withy the cryo solution, seal the membrane and let it equilibrate before looping the crystal. Also I think it better to change the reservoir solution in small steps (like from 30% dioxane -- 25% dioxane plus 5%EG -- 20%dioxane plus 10%EG ... until dioxane is 0%). To be honest I never tried it with a dioxane-containing condition, but it worked pretty well with a condition containing 20% isopropanol.
Re: [ccp4bb] Control the crystallization process in the presence of small volatile organic molecules
Chen, Dioxane is not easy to work with, exactly for the reasons you describe. There is one thing you did not mention, which I know to be an additional issue: the quality of the dioxane. I do not know if you need good quality (whatever that is) but it is a fact that crystallization works with dioxane from some manufacturer/lots and not with others. I have never figured out why this is so. For a paper on kinetics and reservoir volume discussion, I would read the work by Forsythe et al (http://journals.iucr.org/d/issues/2002/10/01/ic0013/vidsup.html, doi:10.1107/S0907444902014208) Basically you will find that this paper says that well volume does not matter much. Content of your drop matters a lot. In your case, having a very volatile component, equilibration would be really fast. One way to get away from all that is to do batch experiments, where there is no active transport from a drop into a well. But your question remains very good: you need an oil (?) that will not permit water or a volatile organic substance to escape. I am not sure what would do the trick. Dioxane is a decent cryo-protectant by itself. I would think about adding gooey things like low molecular weight PEG to try to get the drops better behaved. Evaporation of the dioxane will still be an issue, but with a higher viscosity you may not have to chase your crystals through the drop. Hope this helps a little. Mark -Original Message- From: Chen Zhao c.z...@yale.edu To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Wed, Jan 22, 2014 11:00 pm Subject: [ccp4bb] Control the crystallization process in the presence of small volatile organic molecules Dear all, I am now optimizing a hit which contains about 30% 1,4-dioxane using hanging-drop vapor diffusion at 25 degree. I am having a hard time to reproduce the results: most of the times the drops are either dry in one day or full of precipitate, and only occasionally could I get small crystals. Is there a way to control the vapor diffusion process, like using oil to seal the reservoir? (I know paraffin is permeable to dioxane) Also if someone could refer me to studies on the effects of reservoir volume and surface area to the crystallization kinetics, that would be very helpful. I am also seeking for recommendations for freezing crystals in this condition. What kind of cryoprotectant has a higher chance? Another problem is that when I tried to freeze the crystals, the drop dries super rapidly, and the crystals will dissolve if I add reservoir buffer. But I would assume good cryoprotectant could do the job. On the other hand, this points back to my previous question on dioxane-impermeable oil. If this magic oil exists, I could use it to seal the drop when freezing. Thank you for help! Sincerely, Chen
Re: [ccp4bb] Control the crystallization process in the presence of small volatile organic molecules
Dear Mark, Thank you so much for your reply. Interestingly, after several days I posted this question, I found both my previous crystals and precipitant completely dissolved, but instead much larger crystals start to appear. However, most of them are intergrown together, only few are single. And I think I have to get the right timing and freeze them soon, otherwise it may dissolve again. For cryoprotectant, I am also thinking of starting from low molecular weight PEG. Hopefully at least I could manage to get one or two crystals out of a drop. Finger crossed... Best, Chen On Fri, Jan 24, 2014 at 6:31 PM, Mark van der Woerd mjvdwo...@netscape.netwrote: Chen, Dioxane is not easy to work with, exactly for the reasons you describe. There is one thing you did not mention, which I know to be an additional issue: the quality of the dioxane. I do not know if you need good quality (whatever that is) but it is a fact that crystallization works with dioxane from some manufacturer/lots and not with others. I have never figured out why this is so. For a paper on kinetics and reservoir volume discussion, I would read the work by Forsythe et al ( http://journals.iucr.org/d/issues/2002/10/01/ic0013/vidsup.html, doi:10.1107/S0907444902014208)http://dx.doi.org/10.1107/S0907444902014208 Basically you will find that this paper says that well volume does not matter much. Content of your drop matters a lot. In your case, having a very volatile component, equilibration would be really fast. One way to get away from all that is to do batch experiments, where there is no active transport from a drop into a well. But your question remains very good: you need an oil (?) that will not permit water or a volatile organic substance to escape. I am not sure what would do the trick. Dioxane is a decent cryo-protectant by itself. I would think about adding gooey things like low molecular weight PEG to try to get the drops better behaved. Evaporation of the dioxane will still be an issue, but with a higher viscosity you may not have to chase your crystals through the drop. Hope this helps a little. Mark -Original Message- From: Chen Zhao c.z...@yale.edu To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Wed, Jan 22, 2014 11:00 pm Subject: [ccp4bb] Control the crystallization process in the presence of small volatile organic molecules Dear all, I am now optimizing a hit which contains about 30% 1,4-dioxane using hanging-drop vapor diffusion at 25 degree. I am having a hard time to reproduce the results: most of the times the drops are either dry in one day or full of precipitate, and only occasionally could I get small crystals. Is there a way to control the vapor diffusion process, like using oil to seal the reservoir? (I know paraffin is permeable to dioxane) Also if someone could refer me to studies on the effects of reservoir volume and surface area to the crystallization kinetics, that would be very helpful. I am also seeking for recommendations for freezing crystals in this condition. What kind of cryoprotectant has a higher chance? Another problem is that when I tried to freeze the crystals, the drop dries super rapidly, and the crystals will dissolve if I add reservoir buffer. But I would assume good cryoprotectant could do the job. On the other hand, this points back to my previous question on dioxane-impermeable oil. If this magic oil exists, I could use it to seal the drop when freezing. Thank you for help! Sincerely, Chen
Re: [ccp4bb] Control the crystallization process in the presence of small volatile organic molecules
Another thing to try when cryoprotecting any crystals grown at room temp using a volatile precipitant is to first transfer the crystal tray to 4 degrees, then quickly add a cold aliquot of a viscous cryoprotectant to the drop. Evaporation is considerably slower at 4 degrees, and I prefer to use glycerol as cryoprotectant because it significantly slows down the random walk of the crystals through the drop. It makes it so much easier to fish crystals out of the drop if they're not moving at lightning speed... Place the tray at 4 degrees about 20-30 minutes prior to opening the drop. Any longer incubation at lower temp can result in extra nucleation or crystal dissolution. BTW, good luck trying to use a really volatile organic as a cryoprotectant. Every few years I try it and the crystals just get blown out of the cryoloop by the nitrogen gas stream. Diana On Jan 24, 2014, at 5:31 PM, Mark van der Woerd mjvdwo...@netscape.netmailto:mjvdwo...@netscape.net wrote: Chen, Dioxane is not easy to work with, exactly for the reasons you describe. There is one thing you did not mention, which I know to be an additional issue: the quality of the dioxane. I do not know if you need good quality (whatever that is) but it is a fact that crystallization works with dioxane from some manufacturer/lots and not with others. I have never figured out why this is so. For a paper on kinetics and reservoir volume discussion, I would read the work by Forsythe et al (http://journals.iucr.org/d/issues/2002/10/01/ic0013/vidsup.html, doi:10.1107/S0907444902014208)http://dx.doi.org/10.1107/S0907444902014208 Basically you will find that this paper says that well volume does not matter much. Content of your drop matters a lot. In your case, having a very volatile component, equilibration would be really fast. One way to get away from all that is to do batch experiments, where there is no active transport from a drop into a well. But your question remains very good: you need an oil (?) that will not permit water or a volatile organic substance to escape. I am not sure what would do the trick. Dioxane is a decent cryo-protectant by itself. I would think about adding gooey things like low molecular weight PEG to try to get the drops better behaved. Evaporation of the dioxane will still be an issue, but with a higher viscosity you may not have to chase your crystals through the drop. Hope this helps a little. Mark -Original Message- From: Chen Zhao c.z...@yale.edumailto:c.z...@yale.edu To: CCP4BB CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Sent: Wed, Jan 22, 2014 11:00 pm Subject: [ccp4bb] Control the crystallization process in the presence of small volatile organic molecules Dear all, I am now optimizing a hit which contains about 30% 1,4-dioxane using hanging-drop vapor diffusion at 25 degree. I am having a hard time to reproduce the results: most of the times the drops are either dry in one day or full of precipitate, and only occasionally could I get small crystals. Is there a way to control the vapor diffusion process, like using oil to seal the reservoir? (I know paraffin is permeable to dioxane) Also if someone could refer me to studies on the effects of reservoir volume and surface area to the crystallization kinetics, that would be very helpful. I am also seeking for recommendations for freezing crystals in this condition. What kind of cryoprotectant has a higher chance? Another problem is that when I tried to freeze the crystals, the drop dries super rapidly, and the crystals will dissolve if I add reservoir buffer. But I would assume good cryoprotectant could do the job. On the other hand, this points back to my previous question on dioxane-impermeable oil. If this magic oil exists, I could use it to seal the drop when freezing. Thank you for help! Sincerely, Chen UT Southwestern Medical Center The future of medicine, today.
[ccp4bb] Control the crystallization process in the presence of small volatile organic molecules
Dear all, I am now optimizing a hit which contains about 30% 1,4-dioxane using hanging-drop vapor diffusion at 25 degree. I am having a hard time to reproduce the results: most of the times the drops are either dry in one day or full of precipitate, and only occasionally could I get small crystals. Is there a way to control the vapor diffusion process, like using oil to seal the reservoir? (I know paraffin is permeable to dioxane) Also if someone could refer me to studies on the effects of reservoir volume and surface area to the crystallization kinetics, that would be very helpful. I am also seeking for recommendations for freezing crystals in this condition. What kind of cryoprotectant has a higher chance? Another problem is that when I tried to freeze the crystals, the drop dries super rapidly, and the crystals will dissolve if I add reservoir buffer. But I would assume good cryoprotectant could do the job. On the other hand, this points back to my previous question on dioxane-impermeable oil. If this magic oil exists, I could use it to seal the drop when freezing. Thank you for help! Sincerely, Chen
