04 March 2015

Way Down in the X-Ray Weeds

So, what I know about the details of crystallography can fit on the head of a pin...a small pin.  You put pure protein in multiwell plates and then do a huge matrix of crystallization conditions until tiny little crystals form.  Big crystals are best, but you can use tiny crystals or seeds, or with recent advances in technology, to actually collect data.  Then, through some wizardry (some sort of inverse transform) you make spots go to electron density, then with will power and what used to be SGI machines, you thread your protein sequence in, et voila a model of the structure.  I typically don't go for methods papers in fields I have almost no clue in, but this one intrigued me.

This paper aims to increase the efficiency of soaking fragments into crystals to take advantage of 3rd generation synchrotrons. These machines/labs/setups/doohickeys use acoustic droplet injection (ADE), which many people may already be aware of.  In this approach, each fragment soaks into a protein crystal either directly on data collection media or on a moving conveyor belt which then delivers the crystals to the X-ray beam.  The source of inefficiency comes from the time required to soak the fragment in to the crystals (for those where the apparatus is inside the X-ray station. I have no idea what that means, but here is a google image search that might give you an idea.)  A second source is the limit of evaporative dehydration during the fragment soak.  

Using the model system lysozyme and thermolysin the identified factors which can increase efficiency: namely smaller crystals can be used to decrease the soak time.  By small crystals, they are talking things that are 100 microns or less.  The authors go on to state that:
These techniques efficiently use fragment chemicals (~2.5 nL per screened condition), protein (~25 nL per screened condition), space (1120 screened conditions per standard shipping Dewar; no limits using a conveyor belt), and synchrotron beam time (less than 1 second/screened condition).  Evaporative dehydration of the protein crystal limits these fragment screening applications to systems where the fragment soak time is not prohibitive. Slow-binding compounds can be screened (without time constraint) in trays using ADE, but will consume significantly more resources such as purified protein and chemical compounds (~1 µl per screened condition). Hence, it is desirable to identify promising cases where the cost-efficient on-micromesh or on-conveyor soaking methods are adequate.
So, what did I really get out of this paper.  I am amazed by the miniaturization and automation that exists in synchrotrons.  It is really amazing. Its good to get and read the literature for another field.  It can be enlightening.  If I am looking at this correctly, they can screen a 1000 fragments with ~1mg of protein.  With that said, how many people screen for fragments in this way?  It seems not to be resource intensive, if you are sitting at a synchrotron.  But, how much does it cost to sit at that synchrotron?  Are the problems called out here something people encounter every day, or is this a "First World problem" for those sitting at synchrotrons.

What really got me was that the majority of the authors are high school students and undergraduates. This emphasizes to me the commoditization of X-ray, and really all services.  There is a very high level of training that goes in to solving the structure; I get that.  But it seems that many of the steps are commmodities, if you will.  When I was at Merck, they had a directive called (in some form) I-C-E: Innovation-Commoditization-Experimentation.  The concept was the highly trained (and highly paid) scientists needed to focus on innovation.  Once innovation was achieved it led to experimentation (figuring out how to run it routinely).  After that it was a commodity and should be outsourced to enable those scientists to go back to innovating.  It makes sense from a business standpoint, but scary from the scientists standpoint.  I am all for full employment for scientists in industry (trust me on this), but outsourcing can co-exist in industry. Look at the growth in providers from 2011 to 2014. Not sure where I am going with this, but food for thought.


Anonymous said...

Unnecessary optimization. The rate determining step in this process (by far) is still the generation of crystals that grow reliably and diffract to high resolution. Except when it's even further upstream with generating the protein in the first place.

Anonymous said...

This will revolutionize the search for drugs that target lysozyme

Anonymous said...

There must be very few techniques that don't work with lysozyme. A sample changer with >1 effector head is probably the easiest way to speed things up now