Protein Crystallization Hits

When I look at different structural biology labs with their different crystallization processes and equipment they employ I see a wide diversity of techniques used and processes installed. At the extreme end of the spectrum there is the individual investigator working on the structure determination of a single or several related proteins at a time. At the other side are the high-throughput crystallography labs, churning through thousands of constructs and producing hundreds of new X-ray structures. Most labs are somewhere in between and have fine-tuned their operation based as a result of the given resources, expertise and available technology.

At one point in this spectrum there is a peculiar transition, akin to a phase change where interesting things happen. Here the one-investigator-one-target model is replaced by a process-management model. Evidently, the success rates of the high-throughput operations are a lot lower than that of smaller labs. On the other hand, the cost /structure has been continuously driven down by the high-throughput model, making it possible to generate X-ray protein structures at well below $100,000.

Why is there this decline in success rates? One reason is that the 'one-size-fits-all' approach of high-throughput structure determination explicitly accepts failures. Some targets are just not compatible with the particular crystallization regime chosen, the latter of which would be typically varied when handled as a single entity. I suspect though that during the transition to high-throughput, researchers manage processes in a more abstract way and something gets lost. For example they typically don't know precisely what is happening to each individual protein in the pipeline. I think that the intimate knowledge of a protein's characteristics in expression and purification does contribute to success in crystallization.

In addition there may also be a motivational issue. You'd do everything in your capacity to grow better crystals from CLO's (Crystalline Looking Objects) because that's the one target you're focusing on (think back to your days as a PhD student). A process manager however would be more likely to triage a weak hit and instead order a new construct or crank the high-throughput machine a little faster and create more output that way.

I wonder if there's a way to combine the advantages of both regimes. Can we bring back this bond that exists between the person carrying out the crystallization experiment and the target protein? Any ideas?

Cheers,

Peter

A while ago I lost a bet to Mark here at Emerald: in my foolishness I had predicted that structure determination programs (molecular replacement, SAD phasing and so forth) could be done on a cell phone within a year. I lost the bet, but Mark was a good sport and shared the bottle with the team here at Emerald (wondering though whether anybody has done the feat in the meantime).

The topic of hand held computing in crystallography is still fascinating, nevertheless. Cell phones are used for so many different things now but I'm still waiting a bona fide crystallography application, though. Anything out there?

The closest to what I have seen in this regard was recently at the 2009 ACA meeting in Toronto, Canada. Mark Warren from the University of Bath had cut a rectangular hole in his poster and managed to stick his iPhone right behind it, showing a "Crystallograhic Movie". 

Well done, Mark!
Peter

The main use of protein structures in pharma and biotech is of course guiding medicinal chemists in their effort to synthesize better compounds to bind to target proteins. To do this, it is necessary to get structures of protein-ligand complexes. In my discussions with industrial crystallographers I sense that these days more than half of all drug discovery projects are supported by X-ray crystallographic efforts. Hence drug discovery efforts can be effected in lead discovery, for instance via fragment-based screening or in the later phase of lead optimization. Both methodologies require the ligand to associate with the protein either before crystallization or after crystallization. The reality is though that the corresponding experiments, co-crystallization or ligand soaking don't always work. There are lots of reasons why they may not work. What's the back-up plan when soaking or co-crystallization experiments fail?
Cross-linking crystals and soaking them with ligands has worked for PDE10 (human phosphodiesterase 10a) - see this reference. Initially only apo PDE10 crystals could be grown. Ligands did not show up in the corresponding X-ray structures, neither after soaking apo crystals with ligands nor by crystallizing PDE10 together with the ligand. Once the PDE Crystals were cross-linked with glutardialdahyde though, and ligands were soaked in, X-ray structures with ligand bound were obtained.

Another trick in our toolbox,

Peter

Sometimes I wonder how the world will look like in a few years and: how will we be crystallizing proteins in 5 years from now? Of course it is impossible to predict new technological breakthroughs. However, I think there are technologies that are in their infancy now that will be widely used in 5 years. That allows us to extrapolate a bit. Here are some trends that point to fewer primary crystallization screening experiments and less protein sample requirements within 5 years time:

1. Better primary crystallization screening regimes. As more systematically generated protein crystallization data becomes available, it will become easier to extract best practices for the 'first pass' round of crystallization screening . This could go either generic (weeding out the duds) or customized for a particular protein. I think the jury is still out there. Maybe Emerald will have a sequence-in screen-out online tool? ("getting there with escreenbuilder", says Mark). Hard to believe though that there will be a reliable sequence-based crystallization cocktail predictor (please prove me wrong!).
2. More Seeding methods: Looks like seeding and seeding-like techniques will make it big. From what I hear, the microseed matrix screening works well in many labs. What about universal nucleants such as laser pulses and grains?
3. More ultra-low volume crystallization setups. My first crystallization experiments that I set up in the 90's were on the scale of 10 ul. Now the volume of typical crystallization experiments are down to 100 to ca. 10 nano liters (check out Emerald's MPCS). While the technology to go pico Liters is there, for now 10 nano Liters seems to form a practical barrier. Less volume makes sense only if you can get enough crystal mass for X-ray diffraction, or you're only interested in crytallizability and not physical crystals. But how much is crystal mass is enough? The requirement for crystal size has continued to decrease, with structures of crystals less than 10 um possible now and possibly routine in 5 years (think NSLS-II ). For now I don't really think lower than 1 nL crystallization volume makes much sense, things become really messy.

Looking forward to re-read this blog post in 2014 (with foot in mouth?),
Peter

Like in most science disciplines, crystallographers and crystallizers do two things well: (i) carry out experiments and (ii) tell a story about their experiments. This personal exchange from scientist to scientist has always been a part of my work that I enjoy a lot.

We're already through more than half of 2009 and there are still many opportunities for such exchanges. Where and when do crystallographers and crystallizers meet to share their stories? Here's a listing with upcoming 2009 conferences and meetings:

25th European Crystallographic Meeting, August 16-21, 2009, Istanbul, Turkey.

16th CCP4 Northern Protein Structure Workshop, 2-4 September 2009 Carlisle, UK.

ELRIG European Laboratory Robotics Interest Group, 7 and 8th September 2009, Liverpool, UK.

AsCA'09 (Joint Conference of the Asian Crystallographic Association and Chinese Crystallography Society), October 22-25, 2009, Beijing, China.

PSDI 2009 Meeting (Protein Structure Determination in Industry), November 8-10, 2009, Basel, Switzerland.

Enjoy your leg room while you can,
Peter