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 > >
