Protein Crystallization Hits

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

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

Below is a slide presentation on protein crystallization that I am planning to show at PepTalk next week (Jan 9th, 2011, Sunday 3 pm in the Hotel Del Coronado, San Diego as part of the PepTalk Protein Science Week). The intention is to introduce workshop attendees to the topic of protein crystallization. 

If you think there's an important 'facette' missing, let me know now - I still have time to update my slide deck.

All the best,

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