Re: [ccp4bb] Aggregated protein for crystallization
I should have been more clear. If your protein is insoluble aggregate, you can use crystal screen results to get an idea of what buffer conditions favor solubility (and hopefully monodispersity). An example is described nicely in Collins et al, Acta Cryst F 61:1035. Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu On Wed, Feb 22, 2012 at 5:54 PM, Bernhard Rupp (Hofkristallrat a.D.) hofkristall...@gmail.com wrote: You might get lucky by setting up crystallization plates, but chances are you won't get very useful information from them, especially if your aggregated protein is soluble. I seem to fail to understand how crystallization plates would give information in the not-special case of protein aggregates NOT being soluble? BR Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu
Re: [ccp4bb] Aggregated protein for crystallization
Not sure if it will be helpful... but my protein is not the most stable protein, in fact, it does aggregate over time (most likely due to its 'sticky' nature). However, I still get crystals. The problem is the crystals are among the gunks and precipitates. Your case might be different since my protein does not elute out at void volume. Perhaps, try work faster? Sometimes protein aggregates over time rather than immediately. Allan Quoting Raji Edayathumangalam r...@brandeis.edu: Hi Folks, As crazy as it sounds, if you have crystallized and managed to solve the structure of a protein from aggregated protein, please could you share your experience. After many constructs, many many expression schemes and after the usual rigmarole of optimization that is also often discussed on ccp4bb (buffers, glycerol, salt concentrations, pH, detergent, additives etc.), I now have a decently expressing truncated construct for my protein (80 kDa) that is pure but aggregated (elutes in the void volume from a Superdex200 column). I am tempted to make a boatload of aggregated protein and set up some crystal trays (after perhaps testing by CD). So I'd like to hear from folks who have been successful in solving structures from aggregates when many many known and tested optimization methods still leave one with aggregated protein. Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- Allan Pang PhD Student G35 Joseph Priestley Building Queen Mary University of London London E1 4NS Phone number: 02078828480 Twitter: @xerophytes
Re: [ccp4bb] Aggregated protein for crystallization
Dear Raji Running a blue-native gel with lanes in the presence and absence of a reducing agent could prove quite informative. DLS could also return a quick result on the particle distribution in your sample. In that case I would measure samples as fractionated from the superdex200 and compare the measurements after centrifuging the same samples at 100k x g for one hour. Best regards Savvas On 22 Feb 2012, at 00:21, Raji Edayathumangalam r...@brandeis.edu wrote: Hi Folks, As crazy as it sounds, if you have crystallized and managed to solve the structure of a protein from aggregated protein, please could you share your experience. After many constructs, many many expression schemes and after the usual rigmarole of optimization that is also often discussed on ccp4bb (buffers, glycerol, salt concentrations, pH, detergent, additives etc.), I now have a decently expressing truncated construct for my protein (80 kDa) that is pure but aggregated (elutes in the void volume from a Superdex200 column). I am tempted to make a boatload of aggregated protein and set up some crystal trays (after perhaps testing by CD). So I'd like to hear from folks who have been successful in solving structures from aggregates when many many known and tested optimization methods still leave one with aggregated protein. Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Aggregated protein for crystallization
some more thoughts, Do a cryo-EM imaging, it will be ideal than DLS. if the particle sizes are uniform i would think your protein in that state might be useful. cheers Padayatti On Tue, Feb 21, 2012 at 6:21 PM, Raji Edayathumangalam r...@brandeis.edu wrote: Hi Folks, As crazy as it sounds, if you have crystallized and managed to solve the structure of a protein from aggregated protein, please could you share your experience. After many constructs, many many expression schemes and after the usual rigmarole of optimization that is also often discussed on ccp4bb (buffers, glycerol, salt concentrations, pH, detergent, additives etc.), I now have a decently expressing truncated construct for my protein (80 kDa) that is pure but aggregated (elutes in the void volume from a Superdex200 column). I am tempted to make a boatload of aggregated protein and set up some crystal trays (after perhaps testing by CD). So I'd like to hear from folks who have been successful in solving structures from aggregates when many many known and tested optimization methods still leave one with aggregated protein. Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- Pius S Padayatti,PhD, Phone: 216-658-4528
[ccp4bb] Fwd: [ccp4bb] Aggregated protein for crystallization
This was meant to Raji, So here it goes to all. -- Forwarded message -- From: Zhang, Zhen zhen_zh...@dfci.harvard.edu Date: Wed, Feb 22, 2012 at 12:15 PM Subject: RE: [ccp4bb] Aggregated protein for crystallization To: Pius Padayatti ppadaya...@gmail.com Hi Pius, I have done exactly that. I have one protein eluted at void volume of S200 column. The MALS experiment estimates more than 100 copies of monomer in the aggregate. Against my belief, the protein crystallized and diffracted to 2.3A and the resolution was improved to 1.6A later. It turns out that the crystallization buffer breaks the aggregate to dimer and crystallized it from there. I used the lower concentration of the crystallization buffer to run the sizing column and the protein was eluted at reasonable elution time for dimer even though the profile looks ugly and the purified protein is not crystallizable any more. I guess the aggregate somehow protects the folding of the protein and releases the protein slowly to the protein crystal in the right buffer condition. So I think you should setup crystallization trials with your aggregate protein. We cannot search hundreds of conditions for running sizing column. So why not let crystallization trials find that for you? Good luck. Zhen Marasco Laboratory Cancer Immunology and AIDS Dana Farber Cancer institute http://www.marascolab.org/ -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Pius Padayatti Sent: Wednesday, February 22, 2012 11:55 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Aggregated protein for crystallization some more thoughts, Do a cryo-EM imaging, it will be ideal than DLS. if the particle sizes are uniform i would think your protein in that state might be useful. cheers Padayatti On Tue, Feb 21, 2012 at 6:21 PM, Raji Edayathumangalam r...@brandeis.edu wrote: Hi Folks, As crazy as it sounds, if you have crystallized and managed to solve the structure of a protein from aggregated protein, please could you share your experience. After many constructs, many many expression schemes and after the usual rigmarole of optimization that is also often discussed on ccp4bb (buffers, glycerol, salt concentrations, pH, detergent, additives etc.), I now have a decently expressing truncated construct for my protein (80 kDa) that is pure but aggregated (elutes in the void volume from a Superdex200 column). I am tempted to make a boatload of aggregated protein and set up some crystal trays (after perhaps testing by CD). So I'd like to hear from folks who have been successful in solving structures from aggregates when many many known and tested optimization methods still leave one with aggregated protein. Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- Pius S Padayatti,PhD, Phone: 216-658-4528 The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail. -- Pius S Padayatti,PhD, Phone: 216-658-4528
Re: [ccp4bb] Aggregated protein for crystallization
If you haven't done so already, I would screen buffer conditions (pH, salt concentration, glycerol, strongly reducing conditions, ligands, detergents) by DLS to see if you can reduce aggregation. You might get lucky by setting up crystallization plates, but chances are you won't get very useful information from them, especially if your aggregated protein is soluble. There are fluorescent dyes sensitive to aggregation state such as ANS (anilinonaphthalene-8-sulfonate) or Nile Red. I have not used them myself and would like to hear if others have found them useful for screening buffer conditions. Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu
Re: [ccp4bb] Aggregated protein for crystallization
You might get lucky by setting up crystallization plates, but chances are you won't get very useful information from them, especially if your aggregated protein is soluble. I seem to fail to understand how crystallization plates would give information in the not-special case of protein aggregates NOT being soluble? BR Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu
[ccp4bb] Aggregated protein for crystallization
Hi Folks, As crazy as it sounds, if you have crystallized and managed to solve the structure of a protein from aggregated protein, please could you share your experience. After many constructs, many many expression schemes and after the usual rigmarole of optimization that is also often discussed on ccp4bb (buffers, glycerol, salt concentrations, pH, detergent, additives etc.), I now have a decently expressing truncated construct for my protein (80 kDa) that is pure but aggregated (elutes in the void volume from a Superdex200 column). I am tempted to make a boatload of aggregated protein and set up some crystal trays (after perhaps testing by CD). So I'd like to hear from folks who have been successful in solving structures from aggregates when many many known and tested optimization methods still leave one with aggregated protein. Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Aggregated protein for crystallization
Hey, it could be that you just have a big oligomer--any support for that in the relevant literature? A 10-mer would probably beat out an s200, no? Do you have any other way to ascertain the oligomeric state? Jacob On Tue, Feb 21, 2012 at 5:21 PM, Raji Edayathumangalam r...@brandeis.edu wrote: Hi Folks, As crazy as it sounds, if you have crystallized and managed to solve the structure of a protein from aggregated protein, please could you share your experience. After many constructs, many many expression schemes and after the usual rigmarole of optimization that is also often discussed on ccp4bb (buffers, glycerol, salt concentrations, pH, detergent, additives etc.), I now have a decently expressing truncated construct for my protein (80 kDa) that is pure but aggregated (elutes in the void volume from a Superdex200 column). I am tempted to make a boatload of aggregated protein and set up some crystal trays (after perhaps testing by CD). So I'd like to hear from folks who have been successful in solving structures from aggregates when many many known and tested optimization methods still leave one with aggregated protein. Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Aggregated protein for crystallization
I probably it depends on whether you've got gunk or a functionally relevant oligomer in that void volume. Is it active? RecA and Rad51 both form open-ended head-to-tail oligomers and yet they still crystallize. = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Original message Date: Tue, 21 Feb 2012 18:21:03 -0500 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Raji Edayathumangalam r...@brandeis.edu) Subject: [ccp4bb] Aggregated protein for crystallization To: CCP4BB@JISCMAIL.AC.UK Hi Folks, As crazy as it sounds, if you have crystallized and managed to solve the structure of a protein from aggregated protein, please could you share your experience. After many constructs, many many expression schemes and after the usual rigmarole of optimization that is also often discussed on ccp4bb (buffers, glycerol, salt concentrations, pH, detergent, additives etc.), I now have a decently expressing truncated construct for my protein (80 kDa) that is pure but aggregated (elutes in the void volume from a Superdex200 column). I am tempted to make a boatload of aggregated protein and set up some crystal trays (after perhaps testing by CD). So I'd like to hear from folks who have been successful in solving structures from aggregates when many many known and tested optimization methods still leave one with aggregated protein. Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Aggregated protein for crystallization
Hi, Your idea does sound really crazy but actually Jacob had made a very good valid point. Question is do you think your aggregate still functioning or not, if not, can you revive them in vitro and how effective is your refolding process if you are going to refold them? You may want to take a look at structures of some bacterial pore-forming proteins. Many of them can be expressed as large inclusion bodies in heterologous host of which we call aggregate. I know it sound fishy but after refolding and proper proteolytic activation, those proteins retain the ability to induce mortality to their host cell as good as the real one. Anyway, good luck with your endeavors! Puey On Tue, Feb 21, 2012 at 3:26 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Hey, it could be that you just have a big oligomer--any support for that in the relevant literature? A 10-mer would probably beat out an s200, no? Do you have any other way to ascertain the oligomeric state? Jacob On Tue, Feb 21, 2012 at 5:21 PM, Raji Edayathumangalam r...@brandeis.edu wrote: Hi Folks, As crazy as it sounds, if you have crystallized and managed to solve the structure of a protein from aggregated protein, please could you share your experience. After many constructs, many many expression schemes and after the usual rigmarole of optimization that is also often discussed on ccp4bb (buffers, glycerol, salt concentrations, pH, detergent, additives etc.), I now have a decently expressing truncated construct for my protein (80 kDa) that is pure but aggregated (elutes in the void volume from a Superdex200 column). I am tempted to make a boatload of aggregated protein and set up some crystal trays (after perhaps testing by CD). So I'd like to hear from folks who have been successful in solving structures from aggregates when many many known and tested optimization methods still leave one with aggregated protein. Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Aggregated protein for crystallization
Well, depends on what 'aggregated' really means. If it implies reasonably weak oligomerization interaction - and it might not be too strong given that the oligomers remain soluble - a chaotropic crystallization agent (on the extreme end certain high salts, consult Hofmeister for chaotropicity) may rip such soluble aggregates apart or at least get them into a conformationally reasonably well defined state. Crystals do appear/transform even from precipitates on occasion. CD will tell you about the (secondary structure) folding state, not the aggregation state, DLS/MALS would give an estimate for and distribution of the aggregation state. With light scattering, you can also do some systematic experiments exploring what might reduce the aggregate size. I think soluble, defined secondary structure, and a lot of it, is already a good sign. BR On Tue, Feb 21, 2012 at 5:21 PM, Raji Edayathumangalam r...@brandeis.edu wrote: Hi Folks, As crazy as it sounds, if you have crystallized and managed to solve the structure of a protein from aggregated protein, please could you share your experience. After many constructs, many many expression schemes and after the usual rigmarole of optimization that is also often discussed on ccp4bb (buffers, glycerol, salt concentrations, pH, detergent, additives etc.), I now have a decently expressing truncated construct for my protein (80 kDa) that is pure but aggregated (elutes in the void volume from a Superdex200 column). I am tempted to make a boatload of aggregated protein and set up some crystal trays (after perhaps testing by CD). So I'd like to hear from folks who have been successful in solving structures from aggregates when many many known and tested optimization methods still leave one with aggregated protein. Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Aggregated protein for crystallization
Hi, Did you try using a different column like Superose 6? This column works well to separate large molecular weight proteins including oligomers. Ideally if your solution is not cloudy (coming out of void volume) those are not aggregates those might be oligomers. HTH, Shya On Tue, Feb 21, 2012 at 6:21 PM, Raji Edayathumangalam r...@brandeis.eduwrote: Hi Folks, As crazy as it sounds, if you have crystallized and managed to solve the structure of a protein from aggregated protein, please could you share your experience. After many constructs, many many expression schemes and after the usual rigmarole of optimization that is also often discussed on ccp4bb (buffers, glycerol, salt concentrations, pH, detergent, additives etc.), I now have a decently expressing truncated construct for my protein (80 kDa) that is pure but aggregated (elutes in the void volume from a Superdex200 column). I am tempted to make a boatload of aggregated protein and set up some crystal trays (after perhaps testing by CD). So I'd like to hear from folks who have been successful in solving structures from aggregates when many many known and tested optimization methods still leave one with aggregated protein. Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
