Or might be worth going back to the drawing board to design more
constructs (and check them around the same conditions), thermostable
homologs etc.. what about reductive methylation, anyone had luck with
membrane proteins?
tommi
Quoting "Ed Pozharski" <epozh...@umaryland.edu>:
Unfortunately, some crystals don't diffract at all. You may want to try
to 100% verify that it's protein either by SDS-PAGE or mass-spec
(100x100x100 micron crystal could contain ~0.5mcg of protein, so you my
need to use silver staining). If it is, I'd consider trying to get
diffracting crystals by
a) dehydration (chances are slim since you have no diffraction at all)
b) microseeding
c) additive screen (SilverBullets?)
d) screening a lot of crystals (how small are the small ones? They may
be good enough for microfocus beamlines and yes, smaller crystals
generally tend to produce better diffraction quality)
e) this one sounds silly, but make sure you are hitting the crystal with
the beam (just shift it up and down a notch and take single shot - I've
seen this "trick" actually working several times)
The list is not comprehensive. But frankly, having non-diffracting
crystals is a poor place to start, these guys waste a lot of time and
often still don't diffract no matter what you try.
Good luck,
Ed.
On Mon, 2010-08-30 at 21:24 -0400, qiangm zhang wrote:
Hi all,
I got a crystal of one membrane protein (~60kD) from Na/K
phosphate condition (see getit_4), then I got the improved crystal
like getit_5 after trying seeding, different detergents, lipids and
additives. But this crystal does not diffract at all, I already tried
Izit staining which shows it is protein crystal (detergent crystal?).
Does anyone have any good suggestions for the optimization of this
membrane protein crystallization? Thank you in advance.
Best regards
Qiangmin Zhang
Biomedical Science Tower 3
Room1034
3501 Fifth Avenue
Pittsburgh, PA 15260
--
张强敏
--
Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
----------------------------------------------
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
------------------------------ / Lao Tse /
--
Tommi Kajander, Ph.D.
Macromolecular X-ray Crystallography
Research Program in Structural Biology and Biophysics
Institute of Biotechnology
P.O. Box 65 (Street address: Viikinkaari 1, 4th floor)
University of Helsinki
FIN-00014 Helsinki, Finland
Tel. +358-9-191 58903
Fax +358-9-191 59940
