Protein Crystallization Hits

Protein researchers of the world: here's your chance to make a lasting impact with information, ideas and comments on the topic of future disruptive proteomics technologies. The NIH has an RFI (Request for Information, NOT-RM-12-015) out, asking you to provide input in to how to accelerate research in disruptive proteomics technologies.

There are simple online forms where you can provide your anonymous feedback on the following topics:

1. MS-based comprehensive protein identification and quantification. Realistic goals and associated challenges for orders-of-magnitude improvements in dynamic range, sensitivity, throughput or cost. Specific areas of instrumentation (e.g., source design/analyzer geometry, coupling with other instrumentation, etc.) more likely to yield disruptive improvements.
2. Non-MS-based comprehensive protein identification and quantification. Opportunities and challenges for other technologies that could in the near or far term approach/exceed MS-based methods with respect to: accuracy, dynamic range, throughput, and cost in analyzing proteomes.
3. Potential benefits and challenges in incorporation of informatics approaches and/or integration of large protein datasets into the development of the proteomic technologies.
4. Potential important impacts of proteomics technology breakthroughs in basic, discovery and translational biomedical research.

This unique opportunity is available until March 26.

          Quick! Disruptive proteomics technologies. The NIH wants to know what you're thinking.

What are you going to get out of this? No immediate grant awards. But your information may be included in planning  future grant funding opportunities that seek to fund disruptive technologies that have occurred in DNA sequencing technologies in the past few years.

And that impact may be larger than any of the papers you have ever published.

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

For all those that are interested in simplifying membrane protein crystallization trials, you may want to check out this paper on the topic of 'simplifying LCP-based crystallization':

Wallace E, Dranow D, Laible PD, Christensen J, & Nollert P (2011). Monoolein lipid phases as incorporation and enrichment materials for membrane protein crystallization. PloS one, 6 (8) PMID: 21909395

Abstract: he crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established methodology in many structural biology laboratories. The standard procedure employed with this methodology requires the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial in-situ enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases the efficiency of identifying productive crystallization parameters. Finally, we provide a model that explains the incorporation of the membrane protein from solution into the lipid phase via a portal lamellar phase.

This figure explains how this new PLI (post LCP formation incorporation [I know...]) method works:

Figure: Dispense lipid first, then add (purple protein solution), incubate a few hours, then add precipitation reagents.

The simpler, the better,

Peter

Over the years there have been a number of Nobel Prizes awarded to scientists that have used their protein crystallization skills to provide unprecedented insight, usually at atomic resolution, into important biological processes. In appreciation of their contribution I had put these 12 crystallization heroes into the Protein Crystallizers Hall of Fame with the crystallization and structure determination of these proteins: Ribosome (2009), Water & Ion Channels (2003), RNA Polymerase (2006), Photosynthetic Reaction Centre (1988), Ion transporters (1997).

Which are the possible Protein Crystallization-related Nobel Prizes this year? What do you think - which of these target areas would you consider for a 2010 Nobel Prize (Chemistry or Medicine)?

Or here if you prefer to vote via your LinkedIn account: 

There are plenty of reasons to select these protein structures since they have provided useful insight into biological function and should therefore be worthy a 2010 Nobel Prize:

Let's keep in mind though, that the Nobel Committee may take a break from structural biology in 2010 and focus on these areas that come to mind:

□ Discoveries of pluripotent stem cells / dentritic cells

□ Technologies: human genomics, sequencing, DNA microarrays

□ Pathways and Drugs: Leptin, DNA metallo intercalators

Now, with the 2010 Nobel Prize announcements coming up next week I'm keeping my fingers crossed for yet another award for this fine scientific craft. The announcements are expected to be made on

Regardless of the outcome next week, it's definitely an occasion that's worth getting a bottle of champagne out of fridge!

Cheers,

Peter

There's so much progress in membrane protein crystallography generally, and in GPCR crystallography in particular, that I've started a new blog, called GPCR blog. I'm planning to cover topics relating to GPCR structural biology via the Emerald BioStructures website (note the URL: http://emeraldbiostructures.com/gpcrblog). For anybody who's interested in experimental GPCR crystallization conditions, these blog posts may be interesting to read: GPCR Crystallization Conditions  and GPCRs of known structure.

This is a good opportunity to attempt a quick look into the future and anticipate what's yet to come in our field. It is indeed amazing to see all the progress that's been made in the field of membrane protein research within the past ten years. When I decided to join the membrane protein structure research field - this was around the mid nineties - I was warned that this is a super high risk field, may derail my dreams of getting a PostDoc position and not land me a job in either academia or industry. And now, years later, there are so many more crystallographic membrane protein structures - getting close to hundred (depending how you count) and there are substantial efforts in the pharma and biotechnology industry to apply these difficult targets to crystallography based ligand discovery and to lead optimization in drug discovery programs. This is similar to what happened in the late 80ies to soluble protein structure research. Our game has changed dramatically since then, hasn't it?

It feels like a time warp looking at all the progress made in membrane protein structural biology.

So, what's next?

To me the next big steps in structural biology are about scale context in time and space. What does that mean?

1. A better understanding (with atomic resolution, of course) of the detailed dynamics within protein molecules as they go about their work. More precisely, an experimental understanding of how the motions of atoms and their bond rotations & translations taking place in the sub-nanosecond timescale create effects that manifest themselves in what we call "biochemical function" at the milli to second timescale (6-9 orders of magnitude scale difference).
2. The integration of structural data from atoms up, to explain the appearance of macroscopic structures such as cells and organisms (again ca. 6 orders of magnitude).

Of course both of these fields rely heavily on computational tools and require a lot of input by experimentalists as well, providing reality checks and help keeping the models grow better.

My 2 ct,
Peter