Protein Crystallization Hits

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:

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 

A while ago Luc posted this useful comment to as a response to the blog post "Stability not required to grow protein crystals and: Ala, Gly & Phe are your friends":

There is already a very useful webserver to help you design mutants with better chances of crystallization, The Surface Entropy reduction Prediction Server. 

 http://nihserver.mbi.ucla.edu/SER/ 

 Reference: Lukasz Goldschmidt, David Cooper, Zygmunt Derewenda, David Eisenberg. (2007).

Toward rational protein crystallization: A Web server for the design of crystallizable protein variants

Protein Science. 16:1569-1576 (2007 Aug). 

There is also a nice review by Derewenda about protein engineering to enhance crystallizability and improve crystal properties. 

Derewenda ZS.

Application of protein engineering to enhance crystallizability and improve crystal properties.

Acta Crystallogr D Biol Crystallogr. 2010 May;66(Pt 5):604-15.

Thanks for this feedback, Luc!

Peter

While the topic of protein crystallization lost out on the 2010 Nobel Prizes, I herewith award the Funniest Protein Crystallization paper ever

to: Terese Berfors for introducing witty humor into a protein crystallization paper while protecting the lives of animals.

This award is based on a 2003 paper where Terese reviews protein crystallization seeding techniques in a 'tips and tricks' - style paper. Specifically she points out how seeding wands can be made from animal hair, such as cat whiskers and horse hail hair. 

Bergfors, T. (2003)

Seeds to crystals  

Journal of Structural Biology, 142 (1), 66-76 DOI: 10.1016/S1047-8477(03)00039-X

Check out the image below, showing a "Scanning electron micrograph of a horse hair tail with an attached crystal". While this is pretty neat, she also shows an image of the back end of a horse: 

and then comes the figure legend (b) where she writes "An abundant source of material for seeding wands can be found at the flexible C-terminus of this animal."

This cracks me up every time I see it.  Thank you so much, Terese for the Endophin spikes your 2003 paper gives me every so often.

The paper ends on a more serious and highly laudable "technical note" where Terese reminds researchers not to partake in any animal cruelty acts but to simply purchase the protein streak seeding devices she recommends:

No need to agonize your cat, cat whiskers can be purchased.

I checked the Charles River website, called up their technical support and have not yet received an order number for cat whiskers yet. Does anybody reading this know where to purchase cat whiskers from?

Thanks,

Peter

Over the past two years I've systematically reduced the amount of printed paper in my office as well as in my private life. The idea behind this effort is that 'going digital' with all my documents will make it simpler to store and quickly find information that I need. This is work in progress where the crucial software elements that stuck are Sync Toy (general file housekeeping), PICASA (automatic archiving of images) and Google Desktop (searches everything I have on my PC; yes - I'm a PC).

The most recent addition to this toolset is Mendeley. This is a PDF file organization tool that's devised specifically for researchers to archive, annotate and share their PDF articles. To say the least, I'm very impressed with Mendeley's utility. Within a short period of time Mendeley has helped me to aggregate and organize all of my (currently 642 and growing library of) PDF documents. The functionality goes well beyond a traditional Reference Manager. Features are here, the ones that I like particularly are:

• making notes within PDFs,
• sharing libraries over the web,
• backing up my own collection of articles,
• cross-talk with Zotero (a Firefox-based reference manager),
• cross-platform compatibility (I have Mendeley installed on Windows 7 and on iPhone/iPod touch, and use it via the web browser interface - works seamlessly so far) and
• search functionality.

Here's a quick primer to Mendeley:

Mendeley Teaching Presentation

Mendeley : the best research tool since streak-seeding?

There's even a social media facette too; here's my Mendeley profile - friend me if you'd like: 

Oh - and did I mention that Mendeley is available for free?

Cheers,

Peter

Congratulations to Robert G. Edwards to receiving the 2010 Nobel Prize for Physiology or Medicine. Robert is widely known for inventing and promoting the in vitro fertilization procedure. In a way he has 'fathered' 4 million people.

Not bad.

And here's a toast to Richard Heck, Ei-ichi Negishi and Akira Suzuki for receiving the 2010 Nobel Prize for Palladium-catalyzed couplings in organic synthesis.

I'll keep my fingers crossed for protein structural biology Nobels in 2011.

Cheers,

Peter