Wow! Unexpected advices! Thank you very much, for addon and Fab! Ed, you are right in both cases: about my name and protease. I think it is good idea to try to screen for crystallization conditions with bME. Mahesh, I know nothing about your object, as well as buffer conditions. May be you would try bME too?
Best wishes, 2013/8/20 Mahesh Lingaraju <mxl1...@psu.edu> > Hi Evgeny > > I do have a few free cysteine residues but i am not sure if this is the > problem in my case, I say that because i have crystallized this protein > with another ligand without any issues and if oxidation of cysteine > residues is a problem, i would imagine that it would have happened in the > case where it crystallized properly. furthermore, i have other hits with > the protein which gives very mozaic and twinned crystals which diffract > till 2.3 Å. I am having lots of trouble processing that data and for the > work i am trying to do i need to be able to produce and reproduce well > diffracting crystals in a robust manner. > My protein elutes as a single peak on an s-200 once i got rid of > aggregates. > Thanks > > Mahesh > > > On Tue, Aug 20, 2013 at 11:09 AM, Evgeny Osipov <e.m.osi...@gmail.com>wrote: > >> It is very similar with my situation: we are trying to crystallize Fab >> fragments. All we got is spherulites. After rMMS-seeding we got better >> shaped crystals with only 5 A resolution. So now I think that this caused >> by free cysteine near Fab-Fc knee. Oxidation of them leads to uncontrolled >> aggregation and I am trying to figure out how to protect free cysteine >> without reduction of disulphide bridges between heavy and light chains. >> So, here is the question: do you have free cys residues and are your >> protein is monodisperse (SE-chromatography or DLS)? >> >> P.S.: I am sorry for any mistakes in my letter because Thunderbird does >> not provide any grammar checking tool. >> >> 20.08.2013 04:13, Mahesh Lingaraju пишет: >> >> Hi Jürgen >> >> you are right, I did not try any major optimization yet. I only tried >> to vary PEG and protein concentration. That did not really improve things >> too much. The protein mostly forms spherulites beyond 25% PEG. I am also >> thinking that these crystals are poorly diffracting/not diffracting as they >> might be growing from sub-microscopic spherulites ? Thanks for the insights >> >> Mahesh >> >> >> On Mon, Aug 19, 2013 at 8:00 PM, Bosch, Juergen <jubo...@jhsph.edu>wrote: >> >>> Well said Petri, >>> >>> also how much PEG3350 do you have in your conditions ? More than 25% ? >>> I'm going after cryo-conditions at this point, you might want to replace >>> your PEG3350 with smaller PEGs or a mixture of PEG400 and PEG3350. >>> Almost sounds as if no optimization of the original conditions was >>> performed yet. >>> >>> Plenty to do for you, also since you have some crystal use them for >>> seeding into your new screens. >>> >>> Jürgen >>> >>> On Aug 19, 2013, at 5:46 PM, Mahesh Lingaraju wrote: >>> >>> Hi Petri >>> >>> They are non-diffracting at the home source and they are cryo cooled. >>> Like david suggested I guess ill try introducing a buffer as my condition >>> does not have a buffer. it is ammonium acetate and PEG 3350. >>> >>> Thanks for the encouragement ! >>> >>> Mahesh >>> >>> >>> On Mon, Aug 19, 2013 at 5:37 PM, Petri Kursula >>> <petri.kurs...@gmail.com>wrote: >>> >>>> Hi, >>>> >>>> non-diffracting on the home source or state-of-the-art synchrotron? >>>> Cryocooled or room-temperature? What happens if you change the buffer but >>>> keep your pH? etc etc... >>>> >>>> For an important project, one should never ever give up. >>>> >>>> Petri >>>> >>>> >>>> --- >>>> Petri Kursula, PhD >>>> project leader, adjunct professor >>>> Department of Biochemistry & Biocenter Oulu, University of Oulu, Finland >>>> Department of Chemistry, University of Hamburg/DESY, Germany >>>> www.biochem.oulu.fi/kursula >>>> www.desy.de/~petri/research <http://www.desy.de/%7Epetri/research> >>>> petri.kurs...@oulu.fi >>>> --- >>>> >>>> >>>> >>>> >>>> >>>> On Aug 19, 2013, at 11:49 PM, Mahesh Lingaraju <mxl1...@psu.edu> >>>> wrote: >>>> >>>> Hello people >>>> >>>> I recently obtained hexagonal rod like crystals (150x50x20 um) which >>>> turned out to be non diffracting. What is the usual convention for cases >>>> like this ? do people usually give up on the condition or still try to >>>> optimize it ? >>>> >>>> The crystals are also not very reproducible. I believe it is because >>>> of ammonium acetate in the condition causing fluctuations in the pH because >>>> of its volatility. Is there any way to work around such a problem ? >>>> >>>> Thanks >>>> >>>> Mahesh >>>> >>>> >>>> >>>> >>> >>> ...................... >>> Jürgen Bosch >>> Johns Hopkins University >>> Bloomberg School of Public Health >>> Department of Biochemistry & Molecular Biology >>> Johns Hopkins Malaria Research Institute >>> 615 North Wolfe Street, W8708 >>> Baltimore, MD 21205 >>> Office: +1-410-614-4742 >>> Lab: +1-410-614-4894 >>> Fax: +1-410-955-2926 >>> http://lupo.jhsph.edu >>> >>> >>> >>> >>> >> >> >> -- >> Eugene Osipov >> Junior Research Scientist >> Laboratory of Enzyme Engineering >> A.N. Bach Institute of Biochemistry >> Russian Academy of Sciences >> Leninsky pr. 33, 119071 Moscow, Russia >> e-mail: e.m.osi...@gmail.com >> >> > -- Eugene Osipov Junior Research Scientist Laboratory of Enzyme Engineering A.N. Bach Institute of Biochemistry Russian Academy of Sciences Leninsky pr. 33, 119071 Moscow, Russia e-mail: e.m.osi...@gmail.com
