Protein Crystallization Hits

Protein crystallization trial prepared in less than 10 min

How long does it take to set up a protein crystallization experiment?

It all depends on the equipment and the state of organization in your lab. Even with modest manual dexterity, setting up a single 96-well tray really shouldn't take more than 10 min. Allow 10 min of preparation time and you're at only 20 min. In fact, without the use of robotics it is possible to process a 40 uL protein sample and prepare a 96-well protein crystallization trial within less than 20 minutes, all things considered. Multiply that by 2 or 4 if you aim to increase coverage of crystallization phase space. Here's a rough schedule for preparing a single tray consisting of 96 x 0.8 uL sitting drop protein crystallization experiments using the vapor diffusion method :

Activity	Time
Dispensing the well-solutions from a 96 well block (i.e. a Wizard III & IV) into a 96-well sitting drop plate (i.e. Clompact Jr. plates) using a 12 channel pipettor = 8 transfer steps	2-3 min
Dispensing 96 x 0.4 uL of protein solution into the crystallization chamber using multiple volume pipettor (a P20 takes only 3 refills)	2-3 min
Transferring 0.4 uL of well solution to the crystallization chamber using a 12 channel pipettor = 8 transfer steps	2-3 min
Sealing of tray by attaching clear adhesive tape	< 1 min
Preparation time: setting up work space, getting tip boxes ready, unpacking source block and crystallization tray, collection of liquids in the source block with a short spin, removal and application of the cap mat.	ca. 10 min (depends on organization in the lab)

Is setting up a single crystallization tray taking you more than 20 min? Maybe it's worthwhile getting those multichannel pipettors and repeating dispensers, or switching from slow hanging drops to fast sitting drops.

Or just clean up the lab and get those utensils that you already have, better organized ? ;)

Cheers, Peter

Protein structures without growing bugs

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:

A. Deniaud, L. Liguori, I. Blesneac, J.L. Lenormand, E. Pebay-Peyroula

Crystallization of the membrane protein hVDAC1 produced in cell-free system

Biochimica et Biophysica Acta (BBA) - Biomembranes, Volume 1798, Issue 8, August 2010, Pages 1540-1546 

Miyazono, K.I., Watanabe, M., Kosinski, J., Ishikawa, K., Kamo, M., Sawasaki, T., Nagata, K., Bujnicki, J.M., Endo, Y., Tanokura, M., and Kobayashi, I. (2007)

Novel protein fold discovered in the PabI family of restriction enzymes

Nucleic Acids Res., 35, 1908-1918.

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:

Emily T.Beebe,Shin-ichiMakino,AkiraNozawa,YukoMatsubara, Ronnie O.Frederick,JohnG.Primm,MichaelA.GorenandBrianG.Fox Robotic large-scaleapplicationofwheat cell-freetranslationtostructuralstudies including membrane proteins

New Biotechnology July 2010

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]

Protein Crystallization Wiki

While searching the web for crystallization info I stumbled across the CCP4 Crystallization Wiki.

Yes I admit, I had not seen this before. The site has not been updated since 2008 it seems, but there is a wealth of info regarding protein crystallization and specifically on these topics:

• 1 Crystal Characterization
• 2 Crystal Imperfections
• 3 Crystal Growth
• 4 Crystal handling
• 5 Data collection
• 6 References

 

CCP4 protein crystallization wiki home page

 

Enjoy,

Peter

 

 

Best protein crystallization blog posts in 2010 so far

Below is a list of the 10 most popular blog posts from the first half of the year (2010):

 

 

Thanks for your all your comments and feedback!

Peter

Are you a Crystallographer?

How do you know who is a crystallographer?

One way to find out is to check  the World Directory of  Crystallographers at http:/wdc.iucr.org/

In her Vol18(2) editorial (not online yet) of the ICUR newsletter Sine Larsen encourages all crystallographers to sign up and make it easier to be known to your colleagues and potential collaborators.

 

Signing up is easy. Not listed yet? Sign up now.

 

If you're not listed in the World Directory of Crystallographers, you don't count.

 

Cheers,

Peter

Current Protein Crystallization Patent Applications

 The US Patent & Trademark Office publishes patent applications one year after their submission. Since the patent granting process usually takes years, the USPTO database gives a unique opportunity to see new technologies that are not yet - or that will never be - awarded actual patent status. Below is a list of currently active patent applications, as provided by  the US Patent and Trademark Office at http://patft.uspto.gov/ when searching for the key words "Protein Crystallization" in the titles of patent applications. Since most readers of this blog are 'of ordinary skill in the art [sic!] to make and use' protein crystallization you may get some inspiration for your own crystallization experiments.

Cheers,

Peter

Results of Search in AppFT Database for:

TTL/"protein crystallization": 14 applications.

Hits 1 through 14 out of 14

 

5 min to Protein Crystallization Condition

The quickest way to find the crystallization condition for a particular protein?

This: BMCD4 .

The Biomolecular Crystallization Database, now in its fourth version (4.02) and supporting better database query features than ever before is the best way to search for crystallization conditions for a particular protein.

You're starting a new protein crystallization project? Working on a protein that's been crystallized before? BMCD4 may give you a head start in successfully establishing crystallization.

Just 5 min spent searching the BMCD4 may save you a month's worth of work in the crystallization lab.

Cheers,

Peter

Plate material a source for ligand-complex crystallization failure?

What a pleasant surprise I had the other week: while sifting through the literature, searching for crystallization cocktails I found a paper that mentioned the Emerald BioSystems Compact Junior plates (available here)

The authors say in the Methods section under "Crystallization...": " Crystals were grown in sitting drops by vapor diffusion using 96-well plates (Emerald BioSystems plate type EBS-XJR)." Thanks for that explicit note! While the crystallization plate can make a substantial  difference (see blog post Crystallization Game Changer Try a Different Plate ) I don't know if is a key to successfully reproducing the crystallization of the Beta Toxin from Staphylococcus aureus. However, the Compact Junior plates are made out of Polypropylene, a plastic material that is very hydrophobic (holds the drops in a nice round shape) and has a very low water permeability and interesting optical properties. This is different from most other protein crystallization plates that are made out of Polystyrene with different material properties.

Related to this topic: while discussing ligand binding assays this week here at Emerald I learned that certain biochemical assays are indeed optimized for plate materials with the notion that some plates may be 'stickier' than others for a particular ligand. Can substantial amounts of hydrophobic ligands diffuse into the plastic and 'disappear' from the crystallization drop? This makes me wonder if co-crystallization experiments in sitting-drop setups that do not yield ligand-bound structures should be troubleshot by changing the plate material, or maybe by switching to conventional hanging drop using glass cover slides. Sounds like a sensible thing to do - is anybody doing this?

Are there any studies or anecdotes in the scientific literature that show a correlation of plate material with ligand/protein co-crystallizations

Let me know if you see any.

Thanks!

Peter

 

GPCR crystallography and what's next?

There's so much progress in membrane protein crystallography generally, and in GPCR crystallography in particular, that I've started a new blog, called GPCR blog. I'm planning to cover topics relating to GPCR structural biology via the Emerald BioStructures website (note the URL: http://emeraldbiostructures.com/gpcrblog). For anybody who's interested in experimental GPCR crystallization conditions, these blog posts may be interesting to read: GPCR Crystallization Conditions  and GPCRs of known structure.

This is a good opportunity to attempt a quick look into the future and anticipate what's yet to come in our field. It is indeed amazing to see all the progress that's been made in the field of membrane protein research within the past ten years. When I decided to join the membrane protein structure research field - this was around the mid nineties - I was warned that this is a super high risk field, may derail my dreams of getting a PostDoc position and not land me a job in either academia or industry. And now, years later, there are so many more crystallographic membrane protein structures - getting close to hundred (depending how you count) and there are substantial efforts in the pharma and biotechnology industry to apply these difficult targets to crystallography based ligand discovery and to lead optimization in drug discovery programs. This is similar to what happened in the late 80ies to soluble protein structure research. Our game has changed dramatically since then, hasn't it?

It feels like a time warp looking at all the progress made in membrane protein structural biology.

So, what's next?

To me the next big steps in structural biology are about scale context in time and space. What does that mean?

1. A better understanding (with atomic resolution, of course) of the detailed dynamics within protein molecules as they go about their work. More precisely, an experimental understanding of how the motions of atoms and their bond rotations & translations taking place in the sub-nanosecond timescale create effects that manifest themselves in what we call "biochemical function" at the milli to second timescale (6-9 orders of magnitude scale difference).
2. The integration of structural data from atoms up, to explain the appearance of macroscopic structures such as cells and organisms (again ca. 6 orders of magnitude).

Of course both of these fields rely heavily on computational tools and require a lot of input by experimentalists as well, providing reality checks and help keeping the models grow better.

My 2 ct,
Peter

8 Practical Tips for Membrane Protein Crystallization

What's important to know when switching from crystallizing soluble proteins to crystallizing membrane proteins? I've compiled a list of points that I've made in the past when attempting to answer this question.

1. Go nano volume: Sample preparation involves the use of solubilizing detergent, and membrane proteins are notoriously unstable - unless you or your biochemist friend has worked out "conditions" (buffer, lipid, additives, temperature...) that keep the membrane protein from aggregating. This is all about getting the biochemistry right and often requires a lot more effort since standard conditions that are typically applied for soluble proteins may not be sufficient to keep the protein sample alive for a period of time that's compatible with crystal formation. Due to sample loss and cost in most cases you'll start with substantially less protein sample volume than what you're used to. Don't even think about uL-sized crystallization experiments.

2. Set up more crystallization experiments: Get used to screening more extensively, preparing more crystallization experiments and geting fewer crystal hits. Compared to soluble proteins, there are more parameters to screen for. This is due to the presence of an additional component, amphiphiles (detergent type, concentration, lipids...) and their complex behavior in solution. This dramatically increases the dimensionality of the already multidimensional protein crystallization phase space.

3. Spend more time at the microscope. The phenomenology of drop content is, how shall I say it without discouraging you, 'richer'. There are separate detergent rich phases that can look like oiled out protein, some phases are turbid and there are detergent crystals devoid of any membrane protein that may get you on the wrong track. Some membrane protein crystals may not even have proper facets.

You see, this is a funner game.

Of the Practical Membrane Protein Crystallization Tips listed here I think these 3 are the most useful:

4. Membrane proteins often require harsh detergents for their extraction out of the native membrane. Often crystals grow better with milder, shorter chain detergents.

5. Try to control detergent concentration (measuring it and reducing it). Often the detergent concentrates with the membrane protein and when low MWCO filters are used for sample concentration.

6. Start with crystallization screens that are rich in PEG as opposed to salts. For example the Ozma series of Emerald BioSystems' crystallization screens.

And finally:

7. Read this "Pedestrian guide to membrane protein crystallization" by Michael Wiener (Methods 34, 364-372, 2004).

8. By all means, explore non-traditional crystallization experiments that have worked for a number of membrane proteins. For example, utilizing bicelles or lipidic cubic phases (see primer here, and the Cubic LCP Kit).

Enjoy,

Peter