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.

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?