Protein Crystallization Hits

This week there are two fascinating stories in Nature Methods each giving us a glimpse of what structural biology might look like in a decade or so. Both papers describe a technical tour de force, shooting jets of micro crystals into the beam of a X-ray free electron laser and collecting X-ray diffraction images.

The first report utilizes recombinant protein (TbCatB) crystals that are grown in Sf9 insect cells. Yes, that's right: protein crystals grown in vivo, no crystallization setups necessary here. 

Koopmann, R., Cupelli, K., Redecke, L., Nass, K., DePonte, D., White, T., Stellato, F., Rehders, D., Liang, M., Andreasson, J., Aquila, A., Bajt, S., Barthelmess, M., Barty, A., Bogan, M., Bostedt, C., Boutet, S., Bozek, J., Caleman, C., Coppola, N., Davidsson, J., Doak, R., Ekeberg, T., Epp, S., Erk, B., Fleckenstein, H., Foucar, L., Graafsma, H., Gumprecht, L., Hajdu, J., Hampton, C., Hartmann, A., Hartmann, R., Hauser, G., Hirsemann, H., Holl, P., Hunter, M., Kassemeyer, S., Kirian, R., Lomb, L., Maia, F., Kimmel, N., Martin, A., Messerschmidt, M., Reich, C., Rolles, D., Rudek, B., Rudenko, A., Schlichting, I., Schulz, J., Seibert, M., Shoeman, R., Sierra, R., Soltau, H., Stern, S., Strüder, L., Timneanu, N., Ullrich, J., Wang, X., Weidenspointner, G., Weierstall, U., Williams, G., Wunderer, C., Fromme, P., Spence, J., Stehle, T., Chapman, H., Betzel, C., & Duszenko, M. (2012). In vivo protein crystallization opens new routes in structural biology Nature Methods, 9 (3), 259-262 DOI: 10.1038/nmeth.1859 

The second paper describes a similar experiment, carried out with small crystals of the Blastochloris viridis photosynthetic reaction center grown within lipidic phases. The resulting images actually resemble conventional X-ray diffraction images with proper Bragg spots, good enough to build a somewhat meager 8.2 Å resolution electron density map.

Johansson LC, Arnlund D, White TA, Katona G, Deponte DP, Weierstall U, Doak RB, Shoeman RL, Lomb L, Malmerberg E, Davidsson J, Nass K, Liang M, Andreasson J, Aquila A, Bajt S, Barthelmess M, Barty A, Bogan MJ, Bostedt C, Bozek JD, Caleman C, Coffee R, Coppola N, Ekeberg T, Epp SW, Erk B, Fleckenstein H, Foucar L, Graafsma H, Gumprecht L, Hajdu J, Hampton CY, Hartmann R, Hartmann A, Hauser G, Hirsemann H, Holl P, Hunter MS, Kassemeyer S, Kimmel N, Kirian RA, Maia FR, Marchesini S, Martin AV, Reich C, Rolles D, Rudek B, Rudenko A, Schlichting I, Schulz J, Seibert MM, Sierra RG, Soltau H, Starodub D, Stellato F, Stern S, Strüder L, Timneanu N, Ullrich J, Wahlgren WY, Wang X, Weidenspointner G, Wunderer C, Fromme P, Chapman HN, Spence JC, & Neutze R (2012). Lipidic phase membrane protein serial femtosecond crystallography. Nature methods PMID: 22286383

Granted, all of this is currently in the proof-of-concept stage - no actual high resolution structure determined yet - but this is how new exciting breakthrough technologies often start out.  I'm wondering how long it will take for these technologies to mature to a state where they produce useful resolution structures and when they will become applicable to 'the rest of us'. Ten years, mid of the century maybe?

No protein crystallization setups, no crystal harvest, no cryo. X-FEL kills the crystallization champ.

This might change our game quite a bit.

Cheers,

Peter

Exciting news that I need to share with you: the growing Emerald adopts a new name and leadership: 

We're Emerald Bio now:

The details of our transformation are given in this news release: Leading Proteomics Company Transitions to Emerald Bio, Chooses Key Leadership to Address Growing Market Opportunities

George Abe, new Chief Executive Officer and Peter Nollert, new Chief Technologist at Emerald Bio

We're looking forward to continuing to supply you with Emerald's protein crystallization screening kits, protein crystallization optimization reagents, stock solutions, protein crystallization plates and with sophisticated laboratory instrumentation such as the Opti Matrix Maker for the production of crystallization optimization matrices and the Protein Maker for parallel protien production. 

There's more to come.

Cheers,

Peter

What would you give if you knew how the crystallizability of your target protein compares to 'what's our there'? There's a lot of talk about stability, crystallizability and their relationship and there are these hand waving arguments about supposedly problematic 'floppy regions' in proteins.

So here's a relevant paper that sheds solid data on this topic:

Price W.N. et al., Understanding the physical properties controlling protein crystallization based on analysis of large-scale experimental data. Nature Biotechnology 27(1), 51-57. 2009

 The authors thoroughly mined crystallization data from NESG (Northeast Structural Genomics Consortium) and, amongst a lot of other interesting results, present evidence for these key findings:
1. Overall thermodynamic stability (thermal melt) is not a good predictor of crystallization success
But here's the good news as well: Higher crystallization propensity is found for:
2. Proteins that form defined dimers and higher-mers (as opposed to monomers or aggregated protein)
We DLS lovers always knew this, of course ;)
I was not satisfied though with this hyperbole that the paper concluded with: "The dominant factor determining protein crystallization outcome is the prevalence of well-ordered surface epitopes capable of mediating stereochemically specific interprotein packing interactions" - to me this sounds like: "if the molecules pack well with each other, they'll form crystals".

Duh - "The dominant factor determining protein crystallization outcome is the prevalence of well-ordered surface epitopes capable of mediating stereochemically specific interprotein packing interactions."

Nevertheless, their notion that Glycine, Alanine and Phenylalanine residues are 'good' for crystallization may serve as a useful guide when designing protein surface mutations to enhance your targets' crystallizability. Methinks that this will form a centerpiece in CAPCE (computer aided protein crystal engineering).

Cheers,

Peter

There's exciting news for medical research in the US: Today President Obama announces $5billion in Recovery Act funding for medical research projects.

Francis Collins estimates that this stimulus will eventually create 50,000 more science jobs in the US.

Good times ahead of us.
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