DDM does not form crystals in aqueous solution. The published phase
diagrams show this. In fact, few of the alkyl glycoside detergents
crystallize if the sugar group is a beta anomer, even in organic
solvents. This is one of the reasons why they are so difficult to
purify by recrystallization.
Addressing your questions:
1. Whenever I see crystals only in conditions containing Mg++
conditions, I think phosphate contamination. Did you use a phosphate
buffer in any of your purification steps. What happens in conditions
with Zn or Cd? Even a hint of phosphate in the presence of Mg++ and
a bit of ammonium sulfate will produce struvite (a type of kidney
stone). These tend to be needle crystals.
2. Dissolve up a couple of drops containing the most crystals in SDS-
PAGE sample buffer and see if the protein is intact. Slow
proteolysis of your protein by contaminating proteases can trim it to
a "crystallizable" form. If it is, just consider truncating your
protein for expression.
3. You seem to have a lot of precipitate around with the crystals.
Is this always so? Your DDM concentration seems a bit low.
Remember that even though you are at ~ 2XCMC, you must have enough
detergent to solubilize the protein. At ~ 2XCMC (0.015% or ~0.3 mM),
half of the detergent is not in micelles. If your protein needs to
bind 40 molecules of detergent to stay soluble, you need 4 mM DDM to
solubilize a 100KD protein at 10 mg/mL (~.1mM protein concentration).
Hope this helps and good luck,
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: [EMAIL PROTECTED]
****************************************************************
On Apr 16, 2008, at 10:08 AM, Van Den Berg, Bert wrote:
Hi Ngo,
your needles actually look like very thin plates. They seem
promising to me. From appearance its impossible to tell whether the
crystals are detergent or not. DDM is apparently known to form
crystals, but I've never really gotten any (DDM is very soluble).
What temperature have you used? 4C would favor forming DDM
crystals. The round crystals are often seen in membrane protein
crystallization. They generally don't diffract, contain a lot of
detergent (maybe all detergent) and are very hard to get better (ie
get sharp edges and/or diffraction).
What I would do at this point is trying to get the needle/plate
crystals better (by using techniques/tricks that are used for
soluble proteins, such as vary Mg2+ salt conc and identity,
temperature, volume/reservoir ratio, protein conc. and even trying
different detergents and/or mixtures including DDM). For now I
would go with the assumption that the crystals are protein.
Good luck!
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: [EMAIL PROTECTED]
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm
From: CCP4 bulletin board on behalf of Ngo Duc Tri
Sent: Wed 4/16/2008 9:48 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Optimization of needle crystals?
Dear experts,
You didn't mention which detergent in the conditions. From the
picture it looks
like crystals of detergent.
The initial buffer contains 50mM Tris 7.7, 100mM NaCl and 0.015% DDM.
I got two forms of crystal: needle and round shape.
The needle crystals grow from conditions containing Mg2+ salt and
high pH.
The round crystal grow from conditions containing Mg2+, high pH and
small PEG.
I already fail to optimize the round shape (by changing the PEG
concentration as well as pH or protein concentration). So that's
the reason why I think about optimization of the needle form.
Here I attach more pictures to show you the needle and the round
crystal. I never applied powder diffraction before so maybe it's
hard for me to confirm it's the crystal of detergent or not.
Thank you very much for all of your helpful suggestion!
My best regards,
TriNgo
PhD Student- Sungkyunkwan University, Korea
