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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 <[log in to unmask]> 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 <[log in to unmask]> 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 <[log in to unmask]> 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
---





On Aug 19, 2013, at 11:49 PM, Mahesh Lingaraju <[log in to unmask]> 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
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Office: +1-410-614-4742
Lab:      +1-410-614-4894
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-- 
Eugene Osipov
Junior Research Scientist
Laboratory of Enzyme Engineering
A.N. Bach Institute of Biochemistry
Russian Academy of Sciences
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e-mail: [log in to unmask]