October 26, 2010 15:30
We were excited to host a UV microscope demo from JANSi here last week. UV images of crystallization setups can be used to distinguish protein from salt crystals and we wanted to see first-hand how much of a difference this particular UV microscope for protein crystal inspection from JANSi makes.
For recent reports on the utility of UV absorption and fluorescence, check out Harindarpal Gill's paper:
Gill, H. (2010). Evaluating the efficacy of tryptophan fluorescence and absorbance as a selection tool for identifying protein crystals Acta Crystallographica Section F Structural Biology and Crystallization Communications, 66 (3), 364-372 DOI: 10.1107/S1744309110002022
where these microscopes are compared: PRS-1000 , UVEX, MUVIS, QDI-2010 UV microscopes from Korima, JANSi, Formulatrix and CRAIC Technologies, respectively. One could use this paper as a 'shopping guide' to identify the instrument that best fits one's particular need.
The fundamental idea is that tryptophan containing proteins (>80% of proteins have Trp) absorb UV (280 nm)and fluoresce (320 nm), while salt crystals do not. Hence, such UV microscopes can be used as a first stage process filter to avoid screening many salt crystals with scarce X-ray beamtime. Having a tool to quickly resolve the salt vs. protein crystal issue is very useful.
So, did it work? Of course it works! Salt crystals appear dark and protein crystals appear bright.
The surprising thing for me though was something different: I re-discovered a crystal that I had seen before but was triaged because (i) it was a solitary object (I'm expecting showers of small needles) and (ii) within the crystallization trial there were many other hits that looked better (usually having many objects within a single setup, some with nicer facets). Now that I know that none of what we have tested with X-rays turned out any viable diffraction, I may actually go back and check out this solitary object:
So, contrary to my expected utility for UV microscopes (namely to decrease the number of objects that need to be screened with X-rays) in this case UV microscopy can be used to identify additional hits that would have otherwise been overlooked. Rather than a filter, UV microscopes have utility in focusing your attention on the likely winners.
I was surprised by that,
December 16, 2009 02:19
Taking a wrong turn while hiking the great outdoors can be the start of a big adventure, but in the protein crystallization lab it usually means a waste of time, especially when dealing with optimizing protein crystallization hits. The critical point is a typical go/no-go decision where the question you need to answer is this: is it worthwhile to follow up on this particular crystallization experiment? The answer is "yes" if there are no other, potentially better crystallization hits and if you're sure that the objects you're looking at are protein crystals, though not yet useful for X-ray diffraction experiments. The result of a positive decision will be committing to an often tedious protein crystal optimization project, possibly weeks of re-purification and preparing numerous small variations of that protein crystallization setup.
At this point there's one very simple observation that you can make to avoid taking a wrong turn: check the reservoir solution for crystals. Any crystals there resemble those in the drop?
Since many crystallization cocktails are formulated with high salt concentrations, just a little dehydration can cause salt crystals to form.
So, if there are crystals in the reservoir you're very likely dealing with salt crystals in your drop and not the sought-after protein crystals. Don't take this turn and keep on searching for a better protein crystallization hit.
Good reasons to curb your enthusiasm: crystals in the reservoir solution of (A) hanging drop protein crystallization experiments or in (B) sitting drop protein crystallization setups are a harbinger of bad news: salt crystals.
June 5, 2009 01:51
"I know it when I see it" - what exactly are we searching for when inspecting protein crystallization experiments? Most researchers would say: "Got to have facets!". I think that's a myth though, since some well-diffraction crystals don't have a well-defined habit but exhibit a sphaerical appearance. Indeed, a crystal I once called 'the pig' (not shown) had a snout and a wound up tail at the other end. It yielded decent X-ray diffraction and a structure that got me on a Science paper. How about that as a criterion to define a crystal? Below you'll find a few weird looking crystals, check out the submarine and the angels' wings.
Send us your pictures of your favorite crystals!