Something else you should always try is room-temperature diffraction.
Preferably in-situ diffraction if you can swing it. This is because you
never know if your crystal was "born ugly", or was once beautiful and
peacefully well-ordered before you began batting it around with a nylon
loop like a cat with a moth whilst the drop was evaporating and then
when you finally snared it you swept it through the air and dunked it
into liquid nitrogen.
Just sayin'. Might be a few caveats in there. If the crystal was "born
ugly", then, yes, find a new crystal form or change the construct, but
if it diffracted well, you need to work on your sample prep.
-James Holton
MAD Scientist
On 8/20/2013 1:45 PM, Eugene Osipov wrote:
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 <mailto: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 <mailto: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 <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 <mailto: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 <mailto:e.m.osi...@gmail.com>
