Re: [ccp4bb] Substitution to glycerol during crystallogenesis
Glycerol is known to be able to reduce nucleation. This might be countered by an increase in protein concentration. Vera, L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011) Practical Use of Glycerol in Protein Crystallization. Cryst. Growth Des. 11: 2755–2762. Enrico. On Tue, 03 Apr 2012 13:49:50 +0200, Toby Longwood toby.longw...@gmail.com wrote: Dear all, My question is related to a sample preparation. I’m working with a complex that can be stabilized with glycerol (at least 10%) during purification. The use of detergents does not help. After purification, the sample is homogeneous (EM) and can be concentrated (3-4mg.mL-1) . I already set up many drops, changing several conditions (pH, salt...) but nothing conclusive appeared. I know that crystallogenesis in presence of glycerol works (Sousa, Acta Cryst (1995), ...) however, because of the aspect of the drops (precipitates that seem close to the nucleation phase), I suspect that the glycerol can be one of the limiting factors of the protocol. Has anybody else been already confronted to the same problem? Does someone know if there is an alternate additive to glycerol? Thanks in advance for suggestions/help With best wishes Toby -- 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/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura 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] Substitution to glycerol during crystallogenesis
Florian Schmitzberger wrote: Dear Toby, I don't think there is a basic problem using glycerol in crystallization. Glycerol will affect the vapour pressure (if it is not present in the well/precipitant solution) and 10 % glycerol is ~ 1.3 molar concentration. During equilibration the drops may increase in volume, decreasing the protein concentration. Thus, when using glycerol I think it is generally beneficial to start with a high protein concentration. Perhaps, you can concentrate your protein sample further. It sounds like you are encouraging the OP to crystallize by reverse vapor diffusion. Unless there is reason to believe that diluting the droplets will push the protein toward saturation (salting-in region?), it would seem better to skip the reservoir, seal the wells and crystallize by batch method. Even more sensible, (as you parenthetically hint) add the same concentration glycerol to the reservoir solution to compensate and crystallize by normal forward vapor diffusion. And if you are lucky you can use the glycerol-containing reservoir solution as artificial mother liquor if you need it for mounting crystals or mixing cryoprotectant solutions. Still, it makes sense to use higher protein or PEG to counteract the tendency of glycerol to increase solubility/decrease nucleation. I have on several occasions observed immediate precipitation upon mixing protein solution (containing glycerol) and precipitant solution; drops then cleared up after a short period of time (and crystals eventually formed). In this case, the crystallization experiment starts in the supersaturated zone, and moves towards an undersaturated concentration, traversing the (metastable) zone where nucleation and crystallization can happen (rather than the other way around, which seems the more traditional approach with crystallization by vapour diffusion). Enrico Stura published a recent article, describing an effect of glycerol on crystallization. Vera,L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011) Practical Use of Glycerol in Protein Crystallization. Cryst. Growth Des. 11 :2755–2762. You could replace glycerol with ethylenglycol or a small molecular weight PEG (e.g. 400), which may also have a stabilizing effect on your complex. Regards, Florian On Apr 3, 2012, at 7:49 AM, Toby Longwood wrote: Dear all, My question is related to a sample preparation. I’m working with a complex that can be stabilized with glycerol (at least 10%) during purification. The use of detergents does not help. After purification, the sample is homogeneous (EM) and can be concentrated (3-4mg.mL-1) . I already set up many drops, changing several conditions (pH, salt...) but nothing conclusive appeared. I know that crystallogenesis in presence of glycerol works (Sousa, Acta Cryst (1995), ...) however, because of the aspect of the drops (precipitates that seem close to the nucleation phase), I suspect that the glycerol can be one of the limiting factors of the protocol. Has anybody else been already confronted to the same problem? Does someone know if there is an alternate additive to glycerol? Thanks in advance for suggestions/help With best wishes Toby
[ccp4bb] Substitution to glycerol during crystallogenesis
Dear all, My question is related to a sample preparation. I’m working with a complex that can be stabilized with glycerol (at least 10%) during purification. The use of detergents does not help. After purification, the sample is homogeneous (EM) and can be concentrated (3-4mg.mL-1) . I already set up many drops, changing several conditions (pH, salt...) but nothing conclusive appeared. I know that crystallogenesis in presence of glycerol works (Sousa, Acta Cryst (1995), ...) however, because of the aspect of the drops (precipitates that seem close to the nucleation phase), I suspect that the glycerol can be one of the limiting factors of the protocol. Has anybody else been already confronted to the same problem? Does someone know if there is an alternate additive to glycerol? Thanks in advance for suggestions/help With best wishes Toby
Re: [ccp4bb] Substitution to glycerol during crystallogenesis
Dear Toby, I don't think there is a basic problem using glycerol in crystallization. Glycerol will affect the vapour pressure (if it is not present in the well/precipitant solution) and 10 % glycerol is ~ 1.3 molar concentration. During equilibration the drops may increase in volume, decreasing the protein concentration. Thus, when using glycerol I think it is generally beneficial to start with a high protein concentration. Perhaps, you can concentrate your protein sample further. I have on several occasions observed immediate precipitation upon mixing protein solution (containing glycerol) and precipitant solution; drops then cleared up after a short period of time (and crystals eventually formed). In this case, the crystallization experiment starts in the supersaturated zone, and moves towards an undersaturated concentration, traversing the (metastable) zone where nucleation and crystallization can happen (rather than the other way around, which seems the more traditional approach with crystallization by vapour diffusion). Enrico Stura published a recent article, describing an effect of glycerol on crystallization. Vera,L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011) Practical Use of Glycerol in Protein Crystallization. Cryst. Growth Des. 11 :2755–2762. You could replace glycerol with ethylenglycol or a small molecular weight PEG (e.g. 400), which may also have a stabilizing effect on your complex. Regards, Florian On Apr 3, 2012, at 7:49 AM, Toby Longwood wrote: Dear all, My question is related to a sample preparation. I’m working with a complex that can be stabilized with glycerol (at least 10%) during purification. The use of detergents does not help. After purification, the sample is homogeneous (EM) and can be concentrated (3-4mg.mL-1) . I already set up many drops, changing several conditions (pH, salt...) but nothing conclusive appeared. I know that crystallogenesis in presence of glycerol works (Sousa, Acta Cryst (1995), ...) however, because of the aspect of the drops (precipitates that seem close to the nucleation phase), I suspect that the glycerol can be one of the limiting factors of the protocol. Has anybody else been already confronted to the same problem? Does someone know if there is an alternate additive to glycerol? Thanks in advance for suggestions/help With best wishes Toby
Re: [ccp4bb] Substitution to glycerol during crystallogenesis
We use ethylene glycol and glycerol mainly to reduce nucleation (or showering of crystals). However, we also found that these two additives may not be interchangeable, that is effects of these reagents were markedly different on crystallization behavior of a particular protein. Debasish From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Florian Schmitzberger Sent: Tuesday, April 03, 2012 11:07 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Substitution to glycerol during crystallogenesis Dear Toby, I don't think there is a basic problem using glycerol in crystallization. Glycerol will affect the vapour pressure (if it is not present in the well/precipitant solution) and 10 % glycerol is ~ 1.3 molar concentration. During equilibration the drops may increase in volume, decreasing the protein concentration. Thus, when using glycerol I think it is generally beneficial to start with a high protein concentration. Perhaps, you can concentrate your protein sample further. I have on several occasions observed immediate precipitation upon mixing protein solution (containing glycerol) and precipitant solution; drops then cleared up after a short period of time (and crystals eventually formed). In this case, the crystallization experiment starts in the supersaturated zone, and moves towards an undersaturated concentration, traversing the (metastable) zone where nucleation and crystallization can happen (rather than the other way around, which seems the more traditional approach with crystallization by vapour diffusion). Enrico Stura published a recent article, describing an effect of glycerol on crystallization. Vera,L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011) Practical Use of Glycerol in Protein Crystallization. Cryst. Growth Des. 11 :2755-2762. You could replace glycerol with ethylenglycol or a small molecular weight PEG (e.g. 400), which may also have a stabilizing effect on your complex. Regards, Florian On Apr 3, 2012, at 7:49 AM, Toby Longwood wrote: Dear all, My question is related to a sample preparation. I'm working with a complex that can be stabilized with glycerol (at least 10%) during purification. The use of detergents does not help. After purification, the sample is homogeneous (EM) and can be concentrated (3-4mg.mL-1) . I already set up many drops, changing several conditions (pH, salt...) but nothing conclusive appeared. I know that crystallogenesis in presence of glycerol works (Sousa, Acta Cryst (1995), ...) however, because of the aspect of the drops (precipitates that seem close to the nucleation phase), I suspect that the glycerol can be one of the limiting factors of the protocol. Has anybody else been already confronted to the same problem? Does someone know if there is an alternate additive to glycerol? Thanks in advance for suggestions/help With best wishes Toby
