Protein Crystallization Hits

This is the continuation of a target agnostic survey of often used protein crystallization reagents, based on data obtained from the Biological Macromolecule Crystallization Database (BMCD ver. 4.03). The question I'm trying to address is: which buffers and salts should you inventory?  

Covering protein crystallization space with PEGs seemed a simple affair: a set of only 12 different Polyethyleneglycols is sufficient to formulate ca. 88% of all PEG-based protein crystallization conditions.  

The situation is much less clear cut for buffers and salts that are relevant to protein crystallization. Shy of half of all protein crystallizations listed in the (BMCD ver. 4.03), 45%, can be carried out with 8 different buffers (see Fig. below). Tris buffer seems to be the champion. I interpret this as a result of investigator bias rather than there being a solid scientific reason for this buffer to play such an important role. My explanation is that neutral pH Tris buffers dominate the lab bench, and researchers take what they find first… If this is in fact true, it could support the notion that the nature of the buffer is of somewhat low importance for many protein crystallizations.

Salts are much more interesting since they  can have a dramatic effect on water properties and protein surface decoration, both affecting the ordered association of protein molecules into a crystal. Ammonium sulfate, the classic protein precipitation reagent is the clear winner. Curiously, several of the salts, such as citrates, phosphates and acetates, - the ones that provide both high ion strength and pH buffer capacity are fairly high ranked.

Popular protein crystallization buffers and salts as extracted from the Biological Macromolecule Crystallization Database (BMCD ver. 4.03)

When it comes to warehousing stock solutions for simple and quick preparation of optimization screens, these buffers: CHES, CAPS, Bicine, Tris, Hepes, Imidazole, Bis-Tris, MES

and these salts: magnesium acetate, lithium nitrate, calcium chloride, zinc acetate, potassium/sodium tartrate, sodium citrate, sodium chloride, sodium phosphate, potassium citrate, magnesium sulfate, lithium chloride, calcium acetate, ammonium phosphate, ammonium sulfate are a good start. 

Supplier, name and the associated number of stock solutions that are required for the production and optimization of protein crystallization hits. How this data was generated: Here at Emerald Bio we produce a lot of sparse matrix screens and we accomplish this with our fleet of Matrix Maker instruments that are instructed from a database of screen definitions. Since we keep track of many crystallization screens  we can identify the number of stock solutions that are used in a number of commercial protein crystallization screens.

In average there are 40 different stocks (+- 22) that are required for these protein crystallization screens. 

That's a lot of stock solutions.

What's the highest resolution protein X-ray crystallographic structure? The current record holder is CRAMBIN, a protein extracted from Abysinian kale, with its crystallographic structure determined at a spectacular 0.48Å resolution. PDB entry 3NIR; this is the corresponding reference:

Schmidt A, Teeter M, Weckert E, & Lamzin VS (2011).

Crystal structure of small protein crambin at 0.48 Å resolution. 

Acta crystallographica. Section F, Structural biology and crystallization communications, 67 (Pt 4), 424-8 PMID: 21505232

Apart from the technical feat and minute structural details seen in the resulting electron density, there are several interesting facets to this record breaking X-ray crystallographic resolution. First of all, the protein is rather small. Some would classify this full length, 46 amino acid residue polypeptide as a peptide rather than a protein. But since it was accepted by the 'Protein Data Bank'  let's count it as a bona fide protein. 

In addition to its small size, internal polypeptide motions are restricted by 3 disulfide bonds, aiding the flawless packing  within the crystal.

Finally, the crystallization conditions for this rather hydrophobic protein are quite harsh:

REMARK 280 CRYSTALLIZATION CONDITIONS: 60% ETHANOL IN WATER, PH 7, VAPOR       
REMARK 280  DIFFUSION, SITTING DROP, TEMPERATURE 293K 

Hard as a rock: Crambin X-ray crystallographic structure resolutions over time, as reported in the PDB.  While it seems that something interesting might happen before 2030, the authors rightly argue that the theoretical X-ray resolution limit for Crambin crystals is in the 0.4 Å range, close to that of small molecules.

Inspiring, isn't it?

Many proteins can be crystallized with the help of the molecular crowding agent PEG  (polyethylene glycol). How many proteins? The Biological Macromolecule Crystallization Database (BMCD ver. 4.03) lists that 46% of all protein crystallization crystallizations contain some sort of PEG (that's 20,179 PEG-containing conditions out of a total of 43,406 listed protein crystallizations).

This begs the question: which of the many different PEGs are most useful? - and therefore ought to be available in every protein crystallization lab? To answer this question we've put together a list with commonly used PEGs  (see Fig below).

Figure: These 12 different polyethylene glycols cover ca. 88% of all PEGs induced crystallization space (as derived from the BMCD 4.03)..

In other words: if you've got stocks for all of these 12 Polyethylene Glycol solutions:

PEG 200

PEG 300

PEG 400

PEG 1000

PEG 3350

PEG 4000

PEG 6000

PEG 8000

PEG 10,000

PEG 20,000

PEG 2000 MME

PEG 5000 MME

you're covering ca. 88% of all PEG-induced protein crystallization conditions (according to the crystallization conditions from the BMCD 4.03).

The pioneers that have discovered this immensely important protein crystallization reagents class are Alex McPherson, A. Brzozowski and S. Tolly.  Their publications  helped lift the science of protein crystallization out of the dark ages:

McPherson A Jr (1976). Crystallization of proteins from polyethylene glycol. The Journal of biological chemistry, 251 (20), 6300-3 PMID: 977570

Brzozowski AM, & Tolley SP (1994). Poly(ethylene) glycol monomethyl ethers - an alternative to poly(ethylene) glycols in protein crystallization. Acta crystallographica. Section D, Biological crystallography, 50 (Pt 4), 466-8 PMID: 15299403

Whenever we get a crystallization hit containing PEG we're standing on the shoulders of these giants.

Cheers,

Peter

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