Protein Crystallization Hits

This week I'll prepare my first Iodide soak ever. While we've determined many protein structures at Emerald BioStructures using anomalous data from iodide soaked crystals (see a previous blog post on this topic), I've never prepared iodide-soaked crystals myself. Being a novice I asked Tom Edwards who is an expert in this methodology. Turns out that he's preparing a webinar on this topic for next week: SAD phasing at rotating anode wavelengths using iodide ions. The goal of course is to obtain phases from anomalous X-ray diffraction data, the key to de-novo crystallographic structure determination. 

Tom Edwards on "SAD phasing at rotating anode wavelengths using iodide ions"

 I asked Tom for his 'standard iodide crystal soaking recipe'. Here is it:

1. prepare a 5 M Sodium Iodide stock and a formulation at 2 x of the crystallization cocktail.

2. Mix iodide to a final concentration of 1 M with the 2 x crystallization cocktail and include the cryo reagent.

3. Transfer a single crystal into 1 uL of 1 M soak and check for crystal damage. If there's no visible damage, test X-ray diffraction. Back down with the iodide concentration (0.75 M, 0.5 M etc.) if the quality of  X-ray diffraction  pattern suffers (mosaicity, resolution, split spots etc.).

4. Harvest, cool, mount, diffract, collect... 

The fun part - such as data treatment - will be covered in Tom's webinar

I'm off to the lab.

Peter

When you think about the 'front end' of a protein crystallization project, E.coli is front and center. These bacteria are used to create designed vector constructs and are the standard vehicle for heterologous protein over expression. However, one could also chemically synthesize the protein from scratch - a notorious example is Lysozyme, the 1.04 Å X-ray structure of which has been obtained from crystals grown from a sample that had been obtained by total chemical synthesis. An earlier example for such a path is the structure of the anti-HIV protein AOP-RANTES. 

Chemical synthesis is a tour de force and not a practicable path for most of us. Now though, protein synthesis without involving a chemical fume hood nor culturing E.coli, or any living organisms for that matter, can be done in a standard lab. There are several reports of crystallographic structures that were produced from crystals that had been grown from protein material obtained in cell-free systems. The protein producing machinery is still of biological origin, though. So technically, living organisms are involved - but the timing of their cultivation and the protein production is uncoupled. This circumvents the need to cultivate cells in your own lab and could be done away with altogether when the DNA is of synthetic origin as well.

Here are two X-ray crystallographic protein structure reports that are based on crystals grown from in-vitro expressed protein:

The case for retiring E.coli and utilizing cell-free systems can be made especially for those proteins that are expressed at low yield or that are toxic to E.coli or any other cells (i.e. DNA modifying enzymes) or when specific labels need to be introduced into the protein. Direct access to the protein synthesis machinery is unique and allows tackling difficult targets, such as membrane proteins. A recent summary of such ongoing research is to be published here:

And of course, one of the reasons I mention this synthetic biology route has to do with the fact that we offer via Emerald BioSystems the wheat-germ based protein expression system. Our partner in Japan, Cell Free Sciences has developed reagents and a sophisticated robot that enables researchers to produce milligram amounts of protein. The robot is called Protemist DTII. All you need to do is load the instrument with target-DNA and reagents, klick a button on the screen and walk away. When you're back after one and a half days the instrument has produced (via transcription, translation and affinity purification) your purified target protein. Pretty convenient, isn't it? The instrument that may be most interesting to protein crystallographers though, is the new Protemist XE, shown below. Its capacity is designed to produce tens of milligrams of protein within a one or two day campaign. 

No living cells involved: ten milligram of GFP produced with the Protemist XE using the wheat germ cell-free expression system.

Drop us a note (sales@emeraldbiosystems.com) if you're interested in more information about this protein production system (and tell Frank that Peter sent you :)

Regards,

Peter

P.S. I just saw this comprehensive review article in Nature Biotech, covering the subject of cell-free protein synthesis for functional and structural analysis of membrane proteins:

Junge F, Haberstock S, Roos C, Stefer S, Proverbio D, Dötsch V, Bernhard F.
Advances in cell-free protein synthesis for the functional and structural analysis of membrane proteins.
N Biotechnol. 2010 Jul 15. [Epub ahead of print]

This week I came across a message from PDBsum letting us know that certain figures and captions of a paper we had published in 2008 (Gerdts et al. (2008). Acta Crystallogr D Biol Crystallogr, 64, 1116-1122.) had been included into PDBsum. I had not visited the PDBsum site before and was at first intrigued and then positively surprised about the wealth of information that was presented on a protein structure (3cxk). This is a high-density, information rich way to get a quick impression on a protein structure and other accessory information.

Figure: Screenshot of the PDBsum entry for 3cxk. The crystallization experiment is nicely referenced in PDB-sum.

What I liked in particular about this presentation is that the crystallization experiment becomes part of the story. Our paper described an earlier, beta version of the MPCS - the plug-based nanovolume microcapillary protein crystallization system. Since the publication of the paper in 2008 the technology has matured substantially (check out the New Product Award 2010 that the PlugMaker has received last week).

Any context that goes beyond just reporting the precipitation reagent helps. Having such exquisite detail available when trying to reproduce protein crystallization experiments is often necessary to build on published research.

Way to go EMBL-EBI!
Peter

SSGCID (Seattle Structural Genomics Center for Infectious Disease) is a NIAID-funded structural genomics effort targeting medically relevant proteins of class A and B pathogens. All the crystallography for SSGCID takes place here at Emerald Biostructures. While most structures of our targets can be solved by Molecular Replacement, a number of structure solutions require experimental phase information.

In order to obtain experimental phases, we recently have had very encouraging successes taking advantage of the strong anomalous signal that iodide has at CuKa-wavelength (f"=6.8e-), which is even stronger than the anomalous signal for selenium at the edge (f"=3.7e-). What was intended to be a test study yielded four new in-house phased structure within a month. As a start for this technique, we used a very bold approach: Crystals were transferred stepwise in a buffer containing up to 1M KI. The crystals were soaked in the high-iodide buffer for about 2 hours and then vitrified. Data were collected on our in-house diffractometer (Rigaku MicroMaxHF007 with a Saturn 944+ detector). Again, in a rather bold approach, we collected 360° of data in 0.25° frames with rather short exposures (~10s). Data were processed with XDS/XSCALE.

In our first case, a plasmid partition protein from Borrelia burgdorferi (BobuA.01478.a, pdb: 3k9g), crystallized in a tetragonal space group using NaCl as the precipitant, which was in two steps partially replaced with KI. A 25-fold redundant data set was collected, which showed an anomalous signal out to 2.25Å resolution. Seeding Phaser_EP with 2 strong sites, a total of 16 sites were eventually found and refined. After density modification with PARROT the electron density was readily interpretable (see picture at the end), and almost the entire model was built with BUCCANEER, which then could be extended with ARP/wARP. Further analyses revealed the importance of data redundancy: 180° of data yielded about the same results as 360°, while 90° of data would not yield the structure quite as readily. Other program packages, such as SHARP + ARP/wARP or PHENIX could also solve this structure easily.

In a second case, a deoxycytidine triphosphate deaminase from Anaplasma phagocytophilum (AnphA.00973.a, pdb 3km3), crystallized in a rhombohedral space group and yielded a 10-fold redundant data set up to 2.1Å resolution. Here, the anomalous signal was not as strong as in the previous case. Phaser_EP was seeded with 13 strong and weak iodide sites, eventually 16 sites were found and refined. Again, almost the entire model could be built automatically.

Within the past month, these two structures and another two structures could be solved using anomalous phase information from iodide. Structure #3 yields very well diffracting however twinned crystals; structure #4 only diffracted to 3Å, however phase combination with a weak molecular replacement solution easily yielded the structure. So far, we have only used rather short soaks with 1M KI. Further tests are planned to optimize for lower iodide concentrations.

These results indicated that iodide anomalous phasing can be used as the first shot for determining a new structure that requires experimental phases. With well diffracting crystals and high symmetry, structures can be built within hours after data collection. This technique has shown to work with a twinned data set; for another case a weak Molecular Replacement solution was of trememdous help. Adavantages of iodide soaks are:

• no growth of Se-Met labelled protein is necessary;
• strong anomalous signal in-house;
• high concentrations of iodide are possible during soaks;
• iodide can occupy both positively charged/polarized and hydrophobic pockets, less of a search than with heavy metals;
• it really works!

experimental electron density after in-house iodide phasing

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This week I came across an interesting article reviewing the application of protein crystallization 'chaperones' in the magazine Drug Discovery World 2009. Chun-wa Chung from Glaxo Smith Kline has given her review the provocative title "will crystallization 'chaperones' make all proteins crystallisable?". "Intriguing", I thought - "what's the deal with this chaperone assisted protein crystallography"?

'Crystallization chaperones' are protein molecules that are combined with target proteins to help the nucleation and growth of crystals. The resulting crystals contain both, the target protein and the molecular 'chaperone'. Chun-wa lists several examples where this technique has worked well, notably, ion channels and GPCRs. The current chaperone repertoire is limited to monoclonal antibodies (mAb), fragments (Fv, Fab) of monoclonal antibodies, single chain antibodies (sFv) and other non-antibody proteins such as DARPINs.

While complexes with these chaperones can be grown like any other hetero-dimeric protein complex, it is a tedious process to generate a suitable binder. The fittingly named company Molecular Partners apparently offers this as a service. But you really need a good reason to go this route. Good reason such as: "high-value drug target that failed in conventional high-throughput screening assays", think GPCR (G-protein coupled receptor). You better know that it's worthwhile the extra effort and the substantial detour to generate a specific molecular chaperone to crystallize a particular target protein. I'd be very interested in seeing a large-scale, side-by-side study to determine which path gives you crystals of sufficient quality faster and with less effort:

A) focus on your target protein at hand; learn the ins and outs of it, generate many constructs and set up a lot of crystallization trials with ligands, or

B) generate a specific binder for your target protein at hand and set up crystallization trials with the binder:target protein complex.

Chaperone. Why should I need one?

In her conclusion Chun-wa answers the initial question with a clear 'no' - crystallization chaperones will not make all proteins crystallisable. However, once the detour and extra effort become less of a burden, these molecular chaperones may be used more commonly than now and help crystallize proteins that have otherwise proven recalcitrant to traditional crystallization. Until then, molecular chaperones will be applied to challenging integral membrane proteins where all other 'tricks of the trade' (I'm so glad she's not invoked the 'art'-word) have failed.

Cheers,
Peter