Tom, I doubt that they are DDM crystals, as all the pure BETA-alkyl-glucosides/maltosides are not known to crystallize. Also they are a bitch to crystallize under normal conditions of detergent purification (hence their high cost and the existence of less pure “solubilization” grade products). However, that being said, crystals and paracrystalline structures are observed with detergents of all types.
First, if you could have bought a batch of DDM with high contamination from alpha-dodecyl-maltoside, so you might get crystals; the alpha anomer of these detergents is surprisingly easy to crystallize. Anomer contamination does vary from prep to prep. Second, detergents and protein-detergent complexes can produce quite frustratingly lovely paracrystalline structures that have the physical behavior of butter (on a warm summer day), a stiff rubbery gel, and anything in-between. But so can legitimate membrane protein crystals. Mounting these “crystals” for diffraction usually gives the kind of diffraction you are seeing. What I have found a simple way of determining this: glutaraldehyde cross-linking. First, set up your crystals WITHOUT Tris buffer, as this will screw things up. When the “crystals" have grown, add enough concentrated glutaraldehyde in the reservoir to reach about 0.2-0.5% (10-20% stocks can be easily purchased), close everything up and wait 2-4 hours. Glutaraldehyde is quite volatile (so be careful around it) and will cross-link most membrane protein crystals. Large crystals will turn yellowish (meaning they contain protein), but more importantly adding buffer with detergent but no precipitant will easily dissolve detergent crystals, but not the cross-linked membrane protein crystals. Good luck, Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: rmgarav...@gmail.com **************************************************************** > On Jun 16, 2017, at 10:23 AM, Thomas Warwick <pax...@nottingham.ac.uk> wrote: > > Hi, all. > > I have obtained some crystals (see the imgur link) from my crystal trials of > a membrane protein and I'm concerned that they may be detergent, DDM. > > The drop condition is: > 0.2M NaCl > 0.1M HEPES @ pH 7.0 > 22% PEG500 MME > > Protein sample contains (GF running buffer): > 0.02M Tris > 0.1M NaCl > 0.03% w/v DDM > with 1mM TCEP-HCl added just prior to the trial. Please note however I > concentrated GF eluent fractions 18-fold prior to the trial with a spin > concentrator whose cutoff was smaller than the relative mass of an empty DDM > micelle (it's a small protein) and therefore the DDM concentration could > theoretically be 0.03% x 18 = 0.54% = 10mM... > > The crystals are fragile and oily and occur in a plethora of drops with > varying visual regularity. I've screened a crystal at a synchrotron and it > exhibited essentially amorphous diffraction. > > Has anybody experienced similar entities forming when laying down crystal > trials of membrane proteins solubilized in DDM? If so do they contain > protein? It's my undestanding that DDM is extremely hard to crystallize. Any > information would be appreciated > > http://imgur.com/a/AsZHs (the crystals) > http://imgur.com/a/m88rn (their diffraction pattern) > > Cheers, > Tom > PhD Student within the Structural Biology Group at the University of > Nottingham. > Email: pax...@nottingham.ac.uk
