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

Most of the detergents that are in use today were available 5 or 10 years ago. Maybe the purity has increased over time, but the list of detergents extracted from successfully crystallized membrane proteins really hasn't seen any substantial additions in the past few years. In the previous blog "Applying the 80/20 rule to membrane protein crystallization pre-screening" I showed a list of detergents and referenced statistics that show that with just 8 detergents (dodecyl maltoside, decyl maltoside, nonyl glucoside, octyl glucoside, LDAO, C12E8 C12E9 and undecylmaltoside) about 80% of the membrane protein crystallization 'landscape' is captured.  If you're not working on beta barrel proteins you may as well remove the polyoxyethylene ethers and do with six detergents only, three of them being maltosides.
Only rarely new detergents are conceived, synthesized and tested with membrane proteins. Pil Seok Chae, Sam Gellman and others have done just that, and more: they've come up with a great new detergent class. This is their recent paper on their new series of detergents, called MNGs (maltose-neopentyl glycol amphiphiles):

Chae, P., Rasmussen, S., Rana, R., Gotfryd, K., Chandra, R., Goren, M., Kruse, A., Nurva, S., Loland, C., Pierre, Y., Drew, D., Popot, J., Picot, D., Fox, B., Guan, L., Gether, U., Byrne, B., Kobilka, B., & Gellman, S. (2010). Maltose–neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins Nature Methods, 7 (12), 1003-1008 DOI: 10.1038/nmeth.1526

The MNG molecule looks like this:

Figure: Welcome to the detergent family, MNGs:  maltose-neopentyl glycol amphiphiles. Reserving a spot in the front row on the detergent shelf in the freezer may be a good idea.

The molecule architecture, with the neopentyl in it center, was deliberately designed to provide a more rigid environment for membrane protein crystallization. Looking at the list of preferred detergents, the choice of maltosides (sic!) as a head group seems to be a non-brainer  (this is in retrospect, mind you), but the concept of a quarternary carbon is unique. The successful applications of this new detergent series range from stabilization (activity of beta 2 adrenergic receptor after detergent exchange from DDM), thermostability (of melibiose permease),  solubilization (leucine transporter, light harvesting complex and photosynthetic reaction center) and crystallization (cytochrome b6f & b2AR). While an improvement of crystal quality for cytochrome b6f was not seen, the authors mention that MNG-3 aided in improving crystals of agonist bound beta 2 adrenergic receptor. 

Overall the behavior of the MNGs seem to be on par with DDM (dodecyl maltoside), the currently highest ranked detergent in terms of use for successful crystal growth leading to high-resolution X-ray structures.

I raise may glass to Pil Seok and Sam: well done!
Peter

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 

What's important to know when switching from crystallizing soluble proteins to crystallizing membrane proteins? I've compiled a list of points that I've made in the past when attempting to answer this question.

1. Go nano volume: Sample preparation involves the use of solubilizing detergent, and membrane proteins are notoriously unstable - unless you or your biochemist friend has worked out "conditions" (buffer, lipid, additives, temperature...) that keep the membrane protein from aggregating. This is all about getting the biochemistry right and often requires a lot more effort since standard conditions that are typically applied for soluble proteins may not be sufficient to keep the protein sample alive for a period of time that's compatible with crystal formation. Due to sample loss and cost in most cases you'll start with substantially less protein sample volume than what you're used to. Don't even think about uL-sized crystallization experiments.

2. Set up more crystallization experiments: Get used to screening more extensively, preparing more crystallization experiments and geting fewer crystal hits. Compared to soluble proteins, there are more parameters to screen for. This is due to the presence of an additional component, amphiphiles (detergent type, concentration, lipids...) and their complex behavior in solution. This dramatically increases the dimensionality of the already multidimensional protein crystallization phase space.

3. Spend more time at the microscope. The phenomenology of drop content is, how shall I say it without discouraging you, 'richer'. There are separate detergent rich phases that can look like oiled out protein, some phases are turbid and there are detergent crystals devoid of any membrane protein that may get you on the wrong track. Some membrane protein crystals may not even have proper facets.

You see, this is a funner game.

Of the Practical Membrane Protein Crystallization Tips listed here I think these 3 are the most useful:

4. Membrane proteins often require harsh detergents for their extraction out of the native membrane. Often crystals grow better with milder, shorter chain detergents.

5. Try to control detergent concentration (measuring it and reducing it). Often the detergent concentrates with the membrane protein and when low MWCO filters are used for sample concentration.

6. Start with crystallization screens that are rich in PEG as opposed to salts. For example the Ozma series of Emerald BioSystems' crystallization screens.

And finally:

7. Read this "Pedestrian guide to membrane protein crystallization" by Michael Wiener (Methods 34, 364-372, 2004).

8. By all means, explore non-traditional crystallization experiments that have worked for a number of membrane proteins. For example, utilizing bicelles or lipidic cubic phases (see primer here, and the Cubic LCP Kit).

Enjoy,

Peter

The question I get asked most often when the topic comes to crystallization of membrane proteins in lipidic cubic phases (LCP) is: "what screens can I use?" (for the non-specialists: LCP stands for Lipidic Cubic Phases; these are materials that have been used successfully to serve as host matrices for the crystallization of membrane proteins). The answer is: pretty much everything. After all, the precipitants that have been used successfully are not that different from other protein crystallizations (stop reading here if you're not interested in the details).

The question regarding precipitants and additives goes way back when the LCP crystallization methodology was still in its infancy: I'm talking last century here. Due to their utility in the crystallization of hepta-helical membrane proteins, specifically GPCRs, many crystallizers are interested in using additives for LCP-mediated membrane protein crystallization. Turns out that there's a substantial body of published literature that you can take as a guide through this 'lipid swamp'. To make things simple, let's divide up these additives in lipids, detergents, small molecule amphiphiles and the more traditional crystallization reagents, such as organic solvents, polymers and salt:

Lipidic cubic phases can be doped with a variety of amphiphiles, ranging from LIPIDS such as Cholesterol, DOPE, DOPC, DMPC and PLPC to DETERGENTS (dodecyl maltoside, CTAB, beta octyl glucoside, MEGA9, DTACI and Zwittergent) (see fig 1 below).

Figure 1: Lipid and detergent additives that have been added to LCP to modulate the outcome of membrane protein crystallization experiments. These tables and the one below were taken from a chapter in this book: "14. X-Ray Crystallography of Membrane Proteins: Concepts and Applications of Lipidic Mesophases to Three-Dimensional Membrane Protein Crystallization" G protein-coupled receptors, T.Haga and G.Berstein, editors, 2000, CRC press. P. p 365-388, Loewen, M., Chiu, M., Widmer, C., Landau, E.M., Rosenbusch, J.P., Nollert.

The HOST LIPID that forms a cubic phase is likewise a factor that can be varied:

Figure 2: LCP forming lipids: monopalmitolein, monoolein, monovaccenin, monoeicosenoin and a special cyclopropyl derivative of monoolein.

Indeed, the test protein bacteriorhodopsin crystallized well in a variety of lipids and lipid mixtures:

Figure 3. Crystallization in LCP consisting of different lipids and lipid mixtures, including cholesterol.

That's a lot of compounds that can be screened: LCP (consisting of a range of lipids) with and without additives, at a variety of concentrations. At the time this wealth of possibilities felt like opening "Pandora's Box": while we realized that the LCP method is useful for many membrane protein crystallization trials, the number of possible combination of crystallization components seemed daunting. Regardless, there was a lot of excitement to apply the LCP methods to other membrane proteins, such as bovine rhodopsin or the beta-2-adrenergic receptor.

There's nothing fundamental really, that has changed since these early days. The main progress we've seen in the last 10 years was miniaturization, possibly automation, and setup formats that give better visibility to the crystals.
Foremost, however are more facile protein engineering capabilities and the fact that monoolein has worked so well as the basis host lipid that have made it so simple to apply the LCP method to any membrane protein crystallization trial.

When it comes to the more conventional crystallization reagents, all standard crystallization reagents that are used for soluble proteins can be applied. Some may not be compatible with lipidic cubic phases at all, but this property may actually cause the embedded membrane protein to crystallize (think sponge phases).

Figure 4. Effect of water soluble compounds on cubic phase stability. Polyethylene (PEG) and modified polyethylenes have been used to grow crystals of a variety of membrane proteins. Here is an early paper documenting this:

M. L. Chiu, P. Nollert, M. C. Loewen, H. Belrhali, E. Pebay-Peyroula, J. P. Rosenbusch and E. M. Landau
Crystallization in cubo: general applicability to membrane proteins
Acta Cryst. (2000). D56, 781-784 

Again, all conventional protein crystallization screens can be (and have been) applied to LCP-based membrane protein crystallization. Alternatively, a subset of those conditions that are compatible with the existence of LCP or with protein mobility can be screened using speciality screens, such as Emerald BioSystems' Cubic Screen.

The composition of the crystallization cocktail with all the salts, buffers and additives is described in this Cubic Screen tech-sheet.
There you'll see that alcohols such as ethanol, 2-propanol, 1,4-butanediol, PEGS (400, 1000, 3000, 8000, derivatized PEG 2000 MME), salts (sodium chloride, lithium sulfate, magnesium chloride, zinc acetate, Ammonium sulfate, sodium citrate, Sodium Malonate ) and buffers (Mes, acetate, citrate, hepes, Na/K phosphate, cacodylate, citrate, imidazole) were selected from then exisiting crystallization formulations. To get a variety of concentrations of these precipitation reagents, simple dilution of the entire screen is common practice.

Customers of the Cubic LCP kit have used these components for more than 8 years to screen crystallization conditions and grow membrane protein crystals.

All the best,
Peter

P.S. More on this topic here: Membrane Protein Crystallization In Meso: Lipid Type-Tailoring of the Cubic Phase