Re: [ccp4bb] Crystalization in low PH
Hi, I'm sure there are proteins that were crystallized at low pH but I can't remember which. The best thing is to go to the BMCD database: http://xpdb.nist.gov:8060/BMCD4/index.faces and query it with the key pH (look into advanced search). Cheers, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Sam Arnosti [meisam.nosr...@gmail.com] Sent: Monday, November 07, 2011 7:19 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystalization in low PH Hi everyone I have a protein that is extraordinarily stable at PH=3.0 or even 2.0. I want to crystallize it in the low PH and compare the differences between the crystals in regular PH and low PH. I was wondering how people set up the boxes in low PH, as usual buffers are mostly less acidic. Regards Sam
Re: [ccp4bb] Crystalization in low PH
Tendamistat (1OK0) was crystallized at pH 1.3 and diffracted to 0.93A. George On Mon, Nov 07, 2011 at 05:19:29AM +, Sam Arnosti wrote: Hi everyone I have a protein that is extraordinarily stable at PH=3.0 or even 2.0. I want to crystallize it in the low PH and compare the differences between the crystals in regular PH and low PH. I was wondering how people set up the boxes in low PH, as usual buffers are mostly less acidic. Regards Sam -- Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582
Re: [ccp4bb] Crystalization in low PH
I'm not convinced that you need a conventional buffer at pH 2 or 3. At pH 2, the hydrogen ion concentration is 10 mM. If you want to use something else, the second pKa for sulfuric acid is around 2. The first pKa for phosphoric acid is slightly higher than 2. Lactic acid has a pKa close to 3. Formic acid has a pKa just under 4. Most of these numbers were in an appendix in the first chemistry text you ever used. wink These numbers imply pretty strongly that most crystallization screens emphasizing common salts will require determined modification to hit these low pH values, because many stabilizing anions in the Hoffmeister series will be partially or completely protonated at these pH values. PEG and organic screens will require a smaller hammer to retrofit. On Nov 6, 2011, at 11:19 PM, Sam Arnosti wrote: Hi everyone I have a protein that is extraordinarily stable at PH=3.0 or even 2.0. I want to crystallize it in the low PH and compare the differences between the crystals in regular PH and low PH. I was wondering how people set up the boxes in low PH, as usual buffers are mostly less acidic. Regards Sam
Re: [ccp4bb] Crystalization in low PH
I have crystallized in PEG with citrate at pH 3. If you want to go lower I would suggest maleate: effective pH range pKa 25°Cbuffer 1.2-2.6 1.97 maleate (pK1) 2.2-6.53.13 citrate (pK1) Enrico. On Mon, 07 Nov 2011 14:15:02 +0100, Craig A. Bingman cbing...@biochem.wisc.edu wrote: I'm not convinced that you need a conventional buffer at pH 2 or 3. At pH 2, the hydrogen ion concentration is 10 mM. If you want to use something else, the second pKa for sulfuric acid is around 2. The first pKa for phosphoric acid is slightly higher than 2. Lactic acid has a pKa close to 3. Formic acid has a pKa just under 4. Most of these numbers were in an appendix in the first chemistry text you ever used. wink These numbers imply pretty strongly that most crystallization screens emphasizing common salts will require determined modification to hit these low pH values, because many stabilizing anions in the Hoffmeister series will be partially or completely protonated at these pH values. PEG and organic screens will require a smaller hammer to retrofit. On Nov 6, 2011, at 11:19 PM, Sam Arnosti wrote: Hi everyone I have a protein that is extraordinarily stable at PH=3.0 or even 2.0. I want to crystallize it in the low PH and compare the differences between the crystals in regular PH and low PH. I was wondering how people set up the boxes in low PH, as usual buffers are mostly less acidic. Regards Sam -- 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] Crystalization in low PH
I remembered that people had crystallize a series of streptavidin-2-iminobiotin structures at a low pH. If it might help, check the following PDBIDs: 2RTD 2RTE 2RTI 2RTK 2RTL Hi everyone I have a protein that is extraordinarily stable at PH=3.0 or even 2.0. I want to crystallize it in the low PH and compare the differences between the crystals in regular PH and low PH. I was wondering how people set up the boxes in low PH, as usual buffers are mostly less acidic. Regards Sam
Re: [ccp4bb] Crystalization in low PH
On Mon, 2011-11-07 at 05:19 +, Sam Arnosti wrote: Hi everyone I have a protein that is extraordinarily stable at PH=3.0 or even 2.0. I want to crystallize it in the low PH and compare the differences between the crystals in regular PH and low PH. I was wondering how people set up the boxes in low PH, as usual buffers are mostly less acidic. Regards Sam Not clear if you already have crystals at regular pH, but if you do, you may consider direct transfer to lower pH. Of course, crystals may dissolve, which you could possibly prevent by cross-linking with glutaraldehyde. Three caveats: a) If lattice is incompatible with lower pH, even with cross-linking the resolution may sink to essentially useless levels b) I have no idea if the cross-linking will not be disrupted at really low pH, perhaps someone else can comment on that c) the 3rd reviewer can always say that lattice forces could have prevented a conformational change. But same goes for direct crystallization at low pH (but caries less weight). -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] Crystalization in low PH
Glutaraldehyde works best at low pH On Mon, Nov 7, 2011 at 8:40 AM, Ed Pozharski epozh...@umaryland.edu wrote: On Mon, 2011-11-07 at 05:19 +, Sam Arnosti wrote: Hi everyone I have a protein that is extraordinarily stable at PH=3.0 or even 2.0. I want to crystallize it in the low PH and compare the differences between the crystals in regular PH and low PH. I was wondering how people set up the boxes in low PH, as usual buffers are mostly less acidic. Regards Sam Not clear if you already have crystals at regular pH, but if you do, you may consider direct transfer to lower pH. Of course, crystals may dissolve, which you could possibly prevent by cross-linking with glutaraldehyde. Three caveats: a) If lattice is incompatible with lower pH, even with cross-linking the resolution may sink to essentially useless levels b) I have no idea if the cross-linking will not be disrupted at really low pH, perhaps someone else can comment on that c) the 3rd reviewer can always say that lattice forces could have prevented a conformational change. But same goes for direct crystallization at low pH (but caries less weight). -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
[ccp4bb] Crystalization in low PH
Hi everyone I have a protein that is extraordinarily stable at PH=3.0 or even 2.0. I want to crystallize it in the low PH and compare the differences between the crystals in regular PH and low PH. I was wondering how people set up the boxes in low PH, as usual buffers are mostly less acidic. Regards Sam
