Re: [ccp4bb] pH optimisation for crystallisation
Hello Sreetama: I think for crystallization, everything is hard to say. But if you find your crystal is sensitive to the pH, you certainly can optimize the pH value but it is better not to deviate a lot. For example you can make 0.2 unit interval (for example: pH value 4.5, 4.7, 4.9...etc which are closed to your original pH value ). For the buffer, you can change or not. Another thing is that, you can also incorporate bis-tris in your last purification, since you find your crystal in this buffer. When you do additive screen, the drops which is clear, also can give you important information. You might find a compont which can inhibit crystal formation. You can use it to slow down the crystal formation to get a big or single crystal.However, you see, sometimes, this optimization is time consuming. I suggest you to try seeding. It can give you a big surprise, sometimes. Yu Xiaodi Date: Wed, 8 Feb 2012 11:56:30 +0530 From: somon_...@yahoo.co.in Subject: [ccp4bb] pH optimisation for crystallisation To: CCP4BB@JISCMAIL.AC.UK Dear all, I have a 17 KDa protein that gives crystals in a condition that has 0.1M bis-tris pH 6.5. The crystals are thin needle clusters and do not diffract. I have tried additives, but they haven't improved the crystals. I intend to vary the pH of the condition. My questions are-1. should the buffer be kept the same or can it also be changed (as long as the desired pH is within the range of both the buffers)?2. in case of a different buffer, should its molarity be the same as that of the original one in the crystallization condition? regards,sreetama
Re: [ccp4bb] pH optimisation for crystallisation
If you find yourself in the situation where the buffer you started with is out of range of the pH you would like to attain, there are sets of buffers you can use that contain most of the standard buffers that will give you a fairly linear response across ~4-10, as described by Newman, Acta Cryst D, 2004, v60, pp 610-612. cheers, tom From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Xiaodi Yu [uppsala@hotmail.com] Sent: Wednesday, February 08, 2012 8:06 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] pH optimisation for crystallisation Hello Sreetama: I think for crystallization, everything is hard to say. But if you find your crystal is sensitive to the pH, you certainly can optimize the pH value but it is better not to deviate a lot. For example you can make 0.2 unit interval (for example: pH value 4.5, 4.7, 4.9...etc which are closed to your original pH value ). For the buffer, you can change or not. Another thing is that, you can also incorporate bis-tris in your last purification, since you find your crystal in this buffer. When you do additive screen, the drops which is clear, also can give you important information. You might find a compont which can inhibit crystal formation. You can use it to slow down the crystal formation to get a big or single crystal. However, you see, sometimes, this optimization is time consuming. I suggest you to try seeding. It can give you a big surprise, sometimes. Yu Xiaodi Date: Wed, 8 Feb 2012 11:56:30 +0530 From: somon_...@yahoo.co.in Subject: [ccp4bb] pH optimisation for crystallisation To: CCP4BB@JISCMAIL.AC.UK Dear all, I have a 17 KDa protein that gives crystals in a condition that has 0.1M bis-tris pH 6.5. The crystals are thin needle clusters and do not diffract. I have tried additives, but they haven't improved the crystals. I intend to vary the pH of the condition. My questions are- 1. should the buffer be kept the same or can it also be changed (as long as the desired pH is within the range of both the buffers)? 2. in case of a different buffer, should its molarity be the same as that of the original one in the crystallization condition? regards, sreetama
[ccp4bb] pH optimisation for crystallisation
Dear all, I have a 17 KDa protein that gives crystals in a condition that has 0.1M bis-tris pH 6.5. The crystals are thin needle clusters and do not diffract. I have tried additives, but they haven't improved the crystals. I intend to vary the pH of the condition. My questions are- 1. should the buffer be kept the same or can it also be changed (as long as the desired pH is within the range of both the buffers)? 2. in case of a different buffer, should its molarity be the same as that of the original one in the crystallization condition? regards, sreetama
Re: [ccp4bb] pH optimisation for crystallisation
Dear Sreetama, First of all, there are no hard-and-fast rules for successful crystallisation, try changing as many different variables as possible and go with what works. Having said that, yes, next I would go for a grid optimisation varying the pH in 0.2 or 0.5 units over as wide a range as the buffer can reasonable tolerate at the same molarity, and try different precipitant concentrations on the other axis. If you have enough protein, try plates at different temperatures as well, and different protein concentrations (in multidrop wells you can do this in the same experiment). A very important variable is the protein preparation itself, prepare more protein regularly and try to improve on purity and concentration. Mark Quoting sreetama das: Dear all, I have a 17 KDa protein that gives crystals in a condition that has 0.1M bis-tris pH 6.5. The crystals are thin needle clusters and do not diffract. I have tried additives, but they haven't improved the crystals. I intend to vary the pH of the condition. My questions are- 1. should the buffer be kept the same or can it also be changed (as long as the desired pH is within the range of both the buffers)? 2. in case of a different buffer, should its molarity be the same as that of the original one in the crystallization condition? regards, sreetama Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
