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. 

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

We were excited to host a UV microscope demo from JANSi here last week. UV images of crystallization setups can be used to distinguish protein from salt crystals and we wanted to see first-hand how much of a difference this particular UV microscope for protein crystal inspection from JANSi  makes.

For recent reports on the utility of UV absorption and fluorescence, check out Harindarpal Gill's paper:

where these microscopes are compared: PRS-1000 , UVEX, MUVIS, QDI-2010 UV microscopes from Korima, JANSi, Formulatrix and CRAIC Technologies, respectively. One could use this paper as a 'shopping guide' to identify the instrument that best fits one's particular need.

The fundamental idea is that tryptophan containing proteins (>80% of proteins have Trp) absorb UV (280 nm)and fluoresce (320 nm), while salt crystals do not.  Hence, such UV microscopes can be used as a first stage process filter to avoid screening  many salt crystals with scarce X-ray beamtime. Having a tool to quickly resolve the salt vs. protein crystal issue is very useful.

So, did it work? Of course it works! Salt crystals appear dark and protein crystals appear bright.

The surprising thing for me though was something different: I re-discovered a crystal that I had seen before but was triaged because (i) it was a solitary object (I'm expecting showers of small needles) and (ii) within the crystallization trial there were many other hits that looked better (usually having many objects within a single setup, some with nicer facets). Now that I know that none of what we have tested with X-rays turned out any viable diffraction, I may actually go back and check out this solitary object: 

So, contrary to my expected utility for UV microscopes (namely to decrease the number of objects that need to be screened with X-rays) in this case UV microscopy can be used to identify additional hits that would have otherwise been overlooked. Rather than a filter, UV microscopes have utility in focusing your attention on the likely winners.

I was surprised by that,

Peter 

Over the past two years I've systematically reduced the amount of printed paper in my office as well as in my private life. The idea behind this effort is that 'going digital' with all my documents will make it simpler to store and quickly find information that I need. This is work in progress where the crucial software elements that stuck are Sync Toy (general file housekeeping), PICASA (automatic archiving of images) and Google Desktop (searches everything I have on my PC; yes - I'm a PC).

The most recent addition to this toolset is Mendeley. This is a PDF file organization tool that's devised specifically for researchers to archive, annotate and share their PDF articles. To say the least, I'm very impressed with Mendeley's utility. Within a short period of time Mendeley has helped me to aggregate and organize all of my (currently 642 and growing library of) PDF documents. The functionality goes well beyond a traditional Reference Manager. Features are here, the ones that I like particularly are:

• making notes within PDFs,
• sharing libraries over the web,
• backing up my own collection of articles,
• cross-talk with Zotero (a Firefox-based reference manager),
• cross-platform compatibility (I have Mendeley installed on Windows 7 and on iPhone/iPod touch, and use it via the web browser interface - works seamlessly so far) and
• search functionality.

Here's a quick primer to Mendeley:

Mendeley Teaching Presentation

Mendeley : the best research tool since streak-seeding?

There's even a social media facette too; here's my Mendeley profile - friend me if you'd like: 

Oh - and did I mention that Mendeley is available for free?

Cheers,

Peter

In case you have not heard about Emerald's newest protein crystallization tool, check out the MPCS - the Microcapillary Protein Crystallization System. This is an award winning, simple-to-use crystallization system that enables protein crystal growth with very little protein sample volume requirements (2.5 uL and you're in business). We have recently completed a side-by-side protein crystallization study and report our results in this paper:

This is what the MPCS delivered in terms of successful crystallizations:

1. 28 out of 29 (93%) proteins crystallized as compared to traditional vapor diffusion experiments
2. 90 out of 120 (75%) protein/precipitant combinations lead to initial crystal hits from vapor diffusion experiments
3. Many of the resulting crystals produced high-quality X-ray diffraction data, leading to six novel protein structures that were derived from crystals harvested from MPCS CrystalCards. 

Protein crystallization study using the Plugmaker instrument and Crystal Cards.

The MPCS rocks,

Peter