Protein Crystallization Hits

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

Some of you may know that Emerald BioStructures, as part of the Seattle Structural Genomics Center for Infectious Disease (SSGCID) has contributed to submitting more than 444 protein structures to the PDB in the past 4 years. That's quite an achievement and my congratulations go out to the project teams that are behind these structures, most of them determined via X-ray crystallography. Some of this output, including methods used to achieve this level of productivity, are described in the  September 2011 issue of Acta Cryst F.

One of the protein production methods that has been key for several of my own 2011 protein crystallization projects: lysis scouting with the Protein Maker instrument (described in this open access article "The Protein Maker: an automated system for high-throughput parallel purification". 

Smith, E., Begley, D., Anderson, V., Raymond, A., Haffner, T., Robinson, J., Edwards, T., Duncan, N., Gerdts, C., Mixon, M., Nollert, P., Staker, B., & Stewart, L. (2011). The Protein Maker: an automated system for high-throughput parallel purification Acta Crystallographica Section F Structural Biology and Crystallization Communications, 67 (9), 1015-1021 DOI: 10.1107/S1744309111028776

What is lysis scouting?

Stated simply, lysis scouting combines the testing of a set of cell-lysis buffer conditions with IMAC (ion metal affinity chromatography) . This is done to increase the yield of proteins that appear partially soluble or insoluble under standard lysis buffer conditions.  This procedure results in a clear path forward for scaled-up production of purified protein samples for protein crystallization trials.

How is lysis scouting done?

A single batch of protein expressing E.coli cells is split into 12 pools and lysed by sonication in 12 different buffer conditions. The paper shows as an example P450 51 A1 (CYP51A1) with a 6xHis-Smt tag. This is the outline of the lysis scouting protocol:

1. Prepare 12 aliquots, each corresponding to 3 g of wet cell paste
2. Resuspend in 30 mL lysis buffer (one out of an array of 12) - see table below.

Cell lysis buffers for testing lysis conditions of recombinantly expressed fungal cytochrome P450

3. Sonicate to lyse and spin to remove cell debris

4. Clarify lysates and load on 12 x 1 mL Ni-affinity matrix column

5. Wash, elute and analyze fractions

SDS-PAGE showing that buffers 1C and 1D extract much more of the target protein CYP51A1 (red boxes). L(load), W(wash) and E(elution) fractions are shown next to MW standards.

While well expressed, CYP51A seemed insoluble using standard cell-lysis methods. The lysis-scouting procedure yielded a buffer system with a detergent (CHAPS or octyl glucoside)  in the presence of high salt concentrations (500 mM NaCl).

The utility of the Protein Maker instrument in this process is the short time it takes to run a lysis scouting experiment. Total run time is approximately 1.5 hours (excluding sample analysis). I.e. many proteins can be tested for optimal lysis conditions in a single day - and since the instrument carries out the experiment for you and in parallel, there is plenty of time to strategize the next steps of mg-scale production of the protein sample for crystallization. 

There are many protein structures that we have produced in 2011 that would not exist without Protein Maker supported lysis optimization.

A true work horse.

Peter

Earlier this month I received an email from John Wiley & Sons, confirming that 'The supplement file is now available at http://onlinelibrary.wiley.com/doi/10.1110/ps.073263108/suppinfo'. More than half a year ago I had pointed out that the 'supplementary material' to a paper was not available online. Now it is. Took a while, but  THANKS A LOT for following through, John Wiley & Sons!

When you go to this page, you can download Simon Newstead's alpha-MP-database.xls.

Although somewhat outdated, this spreadsheet contains a concise summary of crystallization conditions from 121 alpha helical membrane protein structures that are listed in the PDB. The extensive analysis of crystallization conditions is described in the accompanying paper:

Newstead S, Ferrandon S, & Iwata S (2008).

Rationalizing alpha-helical membrane protein crystallization.

Protein science, 17 (3), 466-72

PMID: 18218713

While this accounting exercise is not the sexiest of all science, it is of great interest to all of us membrane protein crystallizers . The database is a handy tool that can be consulted when only small quantities of membrane protein sample is available and one is forced to focus on 'what's worked in the past'.  And of course, this data summary is incredibly for the design of new (although highly biased) crystallization matrices. This is exactly what Simon has done: based on his own analysis, a new sparse matrix crystallization screen, called MemGold  is described.

Great work!

Peter

This is the list of answers that I got today from a quick and informal survey that I conducted over 'lunch' with some of our crystallographers: 'what's been the best thing that happened in 2010 in the world of crystallization / crystallography?'

Here we go with the highly opinionated 'Best of 2010 in Protein Crystallography' (in no particular order):

• Iodide and Sulphur SAD phasing catching on big time
• Remote access to GM/CA-CAT & LS-CAT at the Argonne Advanced Photon Source
• Emerald's Plugmaker winning the 2010 Lab Automation New Product Award
• Fabrice Gorrec's Morpheus crystallization screen (it is a 2009 publication, but its impact was felt mostly in 2010)
• The entire 2010 April issue of Acta D on phasing, software tools and best practices

These are my two personal favorites in the 'Best New Software' category: Mendeley (keeping track of ones research PDF collection) and new de-novo feature of the E-Wizard online crystallization screen generator. But where's the 'best 2010 protein crystallization iPhone app?'.

Not directly related to the field of protein crystallography, but this one could serve as an example for us crystallographers:

• The clever choice of the name for the bacteria that have phosphate exchanged by arsenate atoms. Strain GFAJ-1 stands for "Give Felisa A Job" (first author of the paper is Felisa Wolfe-Simon).

Happy 'last days of the decade without a name',

Peter

For an academic researcher paper writing is often the culmination of hundreds, if not many thousands of hours of work. The data is collected, a story is shaping up - how do you go about transmitting your newly found wisdom to your peers?

I have witnessed firsthand several different paper writing styles and practices and am the first to admit that there's no single best way to this. There's some good advice out there, though. For example, Bill Wells suggests in his aptly titled paper "Me Write Pretty One Day: How to Write a Good Scientific Paper"  'a few small steps" to make "...scientific writing clear, straightforward, and digestible". As I'm starting to write a paper myself I find it useful to look at this paper to remind myself of how to write clearly, to define what's my point and then adhere to the 'show, don't tell' rule. Wells guides his readers through 'The Anatomy of a Paper' and gives a lot of good advise on how to tell our story.

As a reader of a protein crystallization paper I expect to see the following:

1. The amino acid sequence of the protein that's expressed.

2. Description of a typical expression and purification experiment. What's the yield & purity? Are there functional assays that provide checkpoints for anybody who wishes to repeat your work?

3. A detailed description of what happened between eluting the protein from the last column and before setting it up for crystallization: temperature(s), storage conditions and time, dialysis (time, volume, MWCO used, volume ratio), concentrating device, centrifugation, filtration, addition of ligands.

4. The crystallization experiment/regime: composition of precipitant solution buffers, crystallization type, volumes, trays, dispensing methodology (stirred or mixed?), screen used, time it took for crystals to show up. Any 'out of the norm' observations, such as 'crystals formed at the air liquid interface'

5. Crystallization result: how many crystals, crystal habit, size, crystal quality (X-ray diffraction limit and any 'Table 1' associated data that resulted from a complete data set). Describe reproducibility (only 1 drop out of 10 produced crystals?, only 1 out of 10 crystals diffracted?) and any insight gained while handling the crystals (did the crystals bend when looping out, do they tend to break? Do they 'melt' when the temperature is changed?).

6. X-ray diffraction experiment: treatment of crystal (cryo used, orientation crystal was mounted) before and during exposure to X-rays. Diffraction equipment used, exposure time, sweep angle.

7. Two images of the crystals: close-up to see the crystal habit and an overview of the entire crystallization experiment. An X-ray image showing diffraction spots (crystal oriented along a special axis would rock).

There's really no good reason to hold back with any such details in a protein crystallization paper (I won't hold it against you when I review your paper). After all, one of the reasons you're writing the crystallization paper is that you aim to provide instructions to a crystallizer who seeks to enable repetition of your work in the future. This is a neat way you can 'pay back' to all scientists that provided you with insightful tips in their papers and that have helped you succeed with your own protein crystallization research project. Pass on the baton.

Cheers, Peter