Protein Crystallization Hits

If I buy a reagent kit for my research, I need to know what's inside. Of course, how else can you carry out an experiment? I can not agree more with Anna Git's complaints in an Nature Comment that there is a lack of transparency imposed by some reagent manufacturers. (see Git, A. (2012). Research tools: A recipe for disaster Nature, 484 (7395), 439-440 DOI: 10.1038/484439a). Here are her grievances:

So here's our stance on this subject: Emerald Bio customers can get all recipe data that goes into any of our reagent kits. Most of this data is supplied in the various tech sheets. For instance, see here for a description of the 96 Wizard III/IV protein crystallization formulations. Should this information not be sufficient for you, call us toll free at 1-888-780-8588 and ask us what else you need. We're glad to share with you more detail on any of our protein crystallization formulations. For instance, recipes for formulations in any of our protein crystallization screens, down to the CAS number if you need, and even raw material supplier information. No need to whisper or sign any confidentiality agreements. 

Why are we doing this? Service to our customers who trust us as a no-formulation secrets manufacturer. And in the long run this openness is in our own interest: Your success in reproducible protein crystallization is our business. 

Thank you for your continued use of our products,

Peter

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.

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

Sorting through my past years' crystallization reports I'm particularly glad about the fact that all of the crystal images are associated with a description of the crystallization precipitation cocktail. In the same image. Of course one can always go back to the original notebook and dig out the particular crystallization condition used, but time tends to cast doubts and having the well solutions directly associated with the crystals shown in  an image is very practical. Here's a simple way to pull out primary screen parameters for association with a particular crystallization experiment:

Let's say the crystallization was done with the JCSG+ screen, formulation from well E4

• Go to E-Screen Builder 
• Select Vendor (Emerald BioSystems),  pick screen: JCSG+
• Click on E4 - et voila, E4 contains 1260 mM Ammonium sulfate, 200 mM Lithium sulfate and 100 mM Tris at pH 8.5.
• From here you can take a screenshot, or use the 'Snipping Tool' to copy and paste this image right next to the image of the crystal in your report.

Finding a particular protein crystallization screen condition is simple: 1. select vendor, 2. select screen, 3. select hit. The crystallization formulation is displayed for you to (4) cut and paste into a crystallization report.

Easy, isn't it?

Peter