Posted by Peter Nollert on Tue, Aug 31, 2010 @ 06:55 AM
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
Posted by Peter Nollert on Tue, Aug 17, 2010 @ 06:00 AM
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:
CCP4 protein crystallization wiki home page
Enjoy,
Peter
Posted by Peter Nollert on Tue, Jul 27, 2010 @ 06:00 AM
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
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PUB. APP. NO.
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Title
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1
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20090218547
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METHODS, COMPOSITIONS, AND KITS FOR PROTEIN CRYSTALLIZATION
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2
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20090015666
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AUTOMATED PROTEIN CRYSTALLIZATION IMAGING
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3
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20080159932
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Protein crystallization method
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4
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20080119642
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CONTROLLED SURFACE TOPOGRAPHY FOR ENHANCED PROTEIN CRYSTALLIZATION RATES
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5
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20080050834
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Protein Crystallization Droplet Actuator, System and Method
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6
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20080044914
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Protein Crystallization Screening and Optimization Droplet Actuators, Systems and Methods
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7
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20070181058
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Protein crystallization apparatus, method of protein crystallization, protein crystallizing agent and process for preparing the same
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8
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20050202405
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Methods, compositions, and kits for protein crystallization
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9
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20050117144
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Automated protein crystallization imaging
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10
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20050075482
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Array for crystallizing protein, device for crystallizing protein and method of screening protein crystallization using the same
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11
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20040138827
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Integrated, intelligent micro-instrumentation platform for protein crystallization
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12
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20030075101
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Protein crystallization in microfluidic structures
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13
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20020183487
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Protein crystallization apparatus and protein crystallization method
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14
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20010027745
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Protein crystallization in microfluidic structures
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Posted by Peter Nollert on Tue, Jun 22, 2010 @ 05:30 AM
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
Posted by Peter Nollert on Tue, Jun 15, 2010 @ 05:00 AM
What would you give if you knew how the crystallizability of your target protein compares to 'what's our there'? There's a lot of talk about stability, crystallizability and their relationship and there are these hand waving arguments about supposedly problematic 'floppy regions' in proteins.
So here's a relevant paper that sheds solid data on this topic:
Price W.N. et al., Understanding the physical properties controlling protein crystallization based on analysis of large-scale experimental data. Nature Biotechnology 27(1), 51-57. 2009
The authors thoroughly mined crystallization data from NESG (Northeast Structural Genomics Consortium) and, amongst a lot of other interesting results, present evidence for these key findings:
1. Overall thermodynamic stability (thermal melt) is not a good predictor of crystallization success
But here's the good news as well: Higher crystallization propensity is found for:
2. Proteins that form defined dimers and higher-mers (as opposed to monomers or aggregated protein)
We DLS lovers always knew this, of course ;)
I was not satisfied though with this hyperbole that the paper concluded with: "The dominant factor determining protein crystallization outcome is the prevalence of well-ordered surface epitopes capable of mediating stereochemically specific interprotein packing interactions" - to me this sounds like: "if the molecules pack well with each other, they'll form crystals".
Duh - "The dominant factor determining protein crystallization outcome is the prevalence of well-ordered surface epitopes capable of mediating stereochemically specific interprotein packing interactions."
Nevertheless, their notion that Glycine, Alanine and Phenylalanine residues are 'good' for crystallization may serve as a useful guide when designing protein surface mutations to enhance your targets' crystallizability. Methinks that this will form a centerpiece in CAPCE (computer aided protein crystal engineering).
Cheers,
Peter
Posted by Peter Nollert on Tue, May 25, 2010 @ 08:00 AM
Kudos to Acta Cryst. F! Acta Cryst F has announced last year that "Tips and Tricks for Structural Biologists" can be published as stand-alone, regular papers under the category "Laboratory Communication".
Finally there is a forum that hosts peer-reviewed crystallization method reports (more in the Editorial ). I can't wait to see highly detailed descriptions of "special methods, equipment modifications, techniques for accomplishing certain tasks etc."
These 'How-to'-type reports should be very useful to all crystallizers practicing the science of protein crystallization. So far there have been several useful reports: one on heavy atom derivatization, two papers on protein crystal UV imaging and one on distinguishing salt vs. protein crystals with a melt test:
T. Beck, C. E. da Cunha and G. M. Sheldrick
How to get the magic triangle and the MAD triangle into your protein crystal
Acta Cryst. (2009). F65, 1068-1070
Synopsis: The handling of the phasing tools I3C and B3C is described, emphasizing practical aspects such as the preparation of solutions and incorporation of the compounds into protein crystals.
K. Dierks, A. Meyer, D. Oberthür, G. Rapp, H. Einspahr and C. Betzel
Efficient UV detection of protein crystals enabled by fluorescence excitation at wavelengths longer than 300 nm
Acta Cryst. (2010). F66, 478-484
Synopsis: Excitation of intrinsic fluorescence at wavelengths longer than 300 nm is effective in the detection of protein crystals in crystallization trials set up in the most commonly used hardware.
H.S. Gill
Evaluating the efficacy of tryptophan fluorescence and absorbance as a selection tool for identifying protein crystals
Acta Cryst. (2010). F66, 364-372
Synopsis: The effectiveness of using ultraviolet microscopes to illuminate protein crystals in high throughput screens is evaluated.
K. Raghunathan, P. T. Harris and D. N. Arvidson
Trial by fire: are the crystals macromolecules?
Acta Cryst. (2010). F66, 615-620
Synopsis: A simple `melt test' to distinguish salt crystals from macromolecule crystals is described.
Protein crystallization tips and tricks announcement taken from the IUCR mag.
Cheers,
Peter
Posted by Peter Nollert on Tue, May 18, 2010 @ 08:30 AM
I've recently re-joined the Faculty of 1000 (Biology). First thing I did: run a search in the discussed papers section, using the keywords "protein crystallization" and filtering for "Tech Advance". Below is the list - quite an eclectic mix. There should be some gold for everyone reading the Crystallization Hits blog. I've printed out 2 of them and put them on my 'papers to read' pile.
Enjoy,
Peter
Posted by Peter Nollert on Tue, May 04, 2010 @ 10:10 AM
There's a lot of lab folk
lore going around in protein crystallization labs. These are stories such as: "
the only thing that every worked for my protein XY is this odd trick" followed by a description of an exotic crystallization trick. Understood, often it is difficult to reconcile what exactly is going on in a particular crystallization experiment. Sometimes, however there's evidence that seemingly weird crystallization tricks do work - check out this example, where
seaweed made the difference. As much as we like to get experimentally tested evidence, in general this rule should hold: '
who crystallizes is right'.
Along these lines, Ivana Tomcova and Ivana Smatanova have come across an interesting case of crystallization dependence that they call 'cross-crystallization'. In their paper
CROSS-CRYSTALLIZATION AS A NEW OPTIMIZATION TOOL OF CRYSTALLIZATION PROCEDURES
Materials Structure, vol. 14, no. 1 (2007)
they describe the crystallization of cytochrome c4 from anaerobic purple sulphur bacterium Thiocapsa roseopersicina. They used the Emerald BioSystems CombiClover Crystallization plate
(thank you very much!) in a special way: all crystallization chambers were filled with a different additive (chloride salts of copper, cadmium, cobalt and barium) and protein was only added to the cupric chloride containing chamber. They optimized this recipe (5 mM CuCl2, ammonium sulfate, citric acid buffer pH5) to a point where the neighboring crystallization chambers were required to contain metal salts (CdCl2, BaCl2, CoCl2), otherwise crystals would not show up.
Fig: Clover Crystallization Plates
There's not much to say other than: apparently it works.
Cheers,
Peter
Posted by Peter Nollert on Tue, Apr 27, 2010 @ 08:00 AM
Membrane protein crystallographers have become very creative in exploring materials that can serve as a matrix for membrane protein crystallization. A few years ago Salem Faham in the lab of James Bowie @ UCLA published a recipe to crystallize bacteriorhodopsin using bicelle preparations. Recently this method received some more attention in the GPCR crystallization field. As a consequence researchers are reading up on these 'old' papers to dig out the protocols for such exotic bicelle-based crystallizations.
Faham S., Bowie, J.
Bicelle Crystallization: A New Method for Crystallizing Membrane Proteins Yields a Monomeric Bacteriorhodopsin Structure
Journal of Molecular Biology, Volume 316 (1), 2002 , pp. 1-6(6)
Since I've dabbled in the 'lipid swamp' myself a little, people sometimes ask me for advice on this topic. The thing is I don't have anything new to contribute, other than having reproduced the bicelle crystallization in the lab. But I'm very happy to see that the FAQ (frequently asked questions) on bicelle based membrane protein crystallization at UCLA is still up.
A great example for openly sharing tips and important technical details that sometimes don't make it into a paper.
Fig: how to make a bicelle sandwich? Check out the FAQ.
Thanks Salem and James for keeping the FAQ site up and running!
Peter
Posted by Peter Nollert on Tue, Apr 20, 2010 @ 06:00 AM
I have to admit, it is somewhat counterintuitive that crystallizers try to crystallize proteins by finding a milieu with low protein solubility by starting with a protein solution and then dilute it. How do you lower solubility by dilution - usually 50:50?. The trick here is of course, that the protein's solubility in the new medium (i.e. the crystallization cocktail that is used to dilute the protein with) is even lower than that in the starting solution. Hence, it seems reasonable to start with a protein buffer that allows to concentrate the protein as high as possible. One way to get to such a buffer is to enhance it for a specific target by increasing the solubility of that target protein. It turns out that this is indeed a practical way to increase the success of crystal screening, mainly due to enhanced nucleation.
Aude Izaac et al. describe a simple procedure to go about such customized buffer selection:
Izaac, A., C.A. Schall and T. C. Mueser (2006)
Assessment of a preliminary solubility screen to improve crystallization trials: uncoupling crystal condition searches
Acta Crystallographica Section D: Biological Crystallography D62, 833-842.
It works by first precipitating (ugh!) the target protein with PEG and then testing its resuspension in a variety of buffers and salts. Here's the protocol :
At first 150 ul of protein solution is precipitated by PEG 8000 (ad 190 ul 40% PEG 8000) at room temperature and spin in aliquots. Then 2 ul of 1 M salt or buffer solutions are added to 18 uL to test if the protein can be re-suspended. After centrifugation (20 ul total vol) the supernatent is tested for protein content. The higher the protein concentration in the sup, the more potent the resolubilization of the buffer for your specific target protein.
In a second step the best salt/buffer combos are determined. The salt and buffers that yielded the largest effect are combined at a standard 100 mM salt and 50 mM buffer concentration and compared to the standard chromatography buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl). Then target protein samples are diluted in the new buffer and concentrated using a 10 kDa MWCO filter. The winner is the buffer that allows the protein to concentrate to the highest level without precipitation.
Fig: What goes up must go down.
Pretty nifty,
Peter
