Weikai,
What you might be experiencing is a detergent effect, i.e., you are
near a detergent-dependent crystallization boundary. We have been hit
with this many times. Under vapor diffusion conditions, sitting or
hanging drop, the protein-detergent complex crystallizes and the free/
bound detergent reaches an equilibrium, but when you open the well,
the drop begins to dry out. Hence, the detergent concentration
increases, which dissolves/cracks the crystals. We also found that
this also happened when our stabilization/freezing buffers had too
high a detergent concentration (lower didn't hurt nearly as badly).
Anecdotally, we have experienced that too high detergent
concentrations inhibited crystallization, perhaps by having too high a
concentration of free detergent micelles, which may interfere with the
crystallization of the protein-detergent complex (there is alway
detergent exchange between the solvent and the crystal). The way we
solved the problem is by setting up the crystals at lower initial
detergent concentrations. Note that as the concentrations of salt and
PEG increases (like during crystallization), the detergent CMC
decreases, even for non-ionic detergents. Therefore, by dropping the
detergent concentration, often to just below the apparent CMC, we
could grow nice stable crystals.
Hope this helps,
Michael
****************************************************************
R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
513 Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824-1319
Office: (517) 355-9724 Lab: (517) 353-9125
FAX: (517) 353-9334 Email: rmgarav...@gmail.com
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On May 24, 2011, at 1:19 PM, weikai wrote:
Hi Folks,
We have some membrane protein crystals that are grown in 30%PEG400,
0.1M Na Citrate pH 4.5, 0.1M LiCl. The protein is purified in DDM.
The crystals are long rods and grown under room temperature in a
hanging drop set up. But once we open the cover slip, we see the
rods start to break and bend in a few seconds. Since it is in high
PEG400, we just directly freeze the crystals. The diffraction only
goes to 10 Ang at synchrotron. Have anybody had similar problem
before and any suggestions?
Thanks a lot,
Weikai
