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
