Just my 2 cents worth, I am with others in that this is actually not unusual. Outside of what Patrick said the first thing to look at if you can is does the crystal diffract at all. You can do this by taking a room temperature shot, the Mitegen loop/sleeve/pins work well for a quick shot or if you have capillaries you can use them too. I have had cryo's kill more crystals then I can remember.
Cheers, Len Thomas ________________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Patrick Loll <pat.l...@drexel.edu> Sent: Wednesday, December 21, 2016 2:53 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal with ZERO diffraction Sadly, I have seen numerous examples of reasonably-sized crystals that give no observable ordered diffraction (I shot a few this weekend, in fact). I can’t give you evidence for what is happening, but I guess that you can build a macroscopic assembly using lattice interactions that are only modestly specific, resulting in a structure that is highly disordered internally, even though it looks OK visually (the jello model). Alternatively, you may have a crystal that was originally well-ordered but subsequently decayed; however, it failed to dissolve because proteins at the surface became cross-linked and held everything together (the soup dumpling model). So no suggestions as how to proceed (except to follow Eddie’s sage advice to grow more/different crystals), but I can assure you that you’re not alone in observing this. Cheers, Pat Loll > On 21 Dec 2016, at 2:44 PM, Tom Huxford <thuxf...@mail.sdsu.edu> wrote: > > Dear ccp4b collective mind and experience, > > Greetings from San Diego. > > I have done my fair share of synchrotron data collection on many diverse > macromolecular crystal systems. But this weekend was the first time that I > ever shot crystals that failed to diffract entirely. > > Details: we have purified and crystallized an ~90 kDa proteolytic fragment > containing a single point mutant version of a myosin motor domain in complex > with a separate light chain polypeptide. The crystals are relatively small > (10-30 microns in each dimension) but clearly crystalline in character (clear > faces, edges, and facets). The crystals tested positive for protein by > absorbance at 280 nm. This weekend we tested more than 40 of them for > diffraction in different cryo solvents and did not observe a single > identifiable diffracted ray. It was as if we had only cryo on the end of our > loops. Increasing the time of exposure or annealing did nothing to improve > the situation. Crystals from a different protein system that we also tested > this weekend on the same beamline diffracted to beyond 1.3 Å. > > I only post this because, in my experience, crystals of this size and > superficial quality always give some signal--even if it is horrifyingly bad. > But never complete diffraction silence. We will work this week to identify > what it is that we "crystallized". But can anybody who has had a similar > experience suggest what it is that could be going on here? > > Thanks in advance for any responses. And happy holidays to us all. > > Tom Huxford. > ====================== > Tom Huxford. > Structural Biochemistry Laboratory > Department of Chemistry & Biochemistry > San Diego State University > (619) 594-1606 >
