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 ?????:
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
<mailto: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 <mailto: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 <http://www.biochem.oulu.fi/kursula>
www.desy.de/~petri/research
<http://www.desy.de/%7Epetri/research>
petri.kurs...@oulu.fi <mailto:petri.kurs...@oulu.fi>
---
On Aug 19, 2013, at 11:49 PM, Mahesh Lingaraju
<mxl1...@psu.edu <mailto: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 <tel:%2B1-410-614-4742>
Lab: +1-410-614-4894 <tel:%2B1-410-614-4894>
Fax: +1-410-955-2926 <tel:%2B1-410-955-2926>
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
