by Peter Nollert
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,
Peter